Abstract
The toxic effects of mercury in earthworms and the potential alleviation effect of hydrogen-rich water (HRW) using ATR-FTIR and LC–MS analysis methods were investigated. Different concentrations of mercury chloride (H1: 5 µg/mL, H2: 10 µg/mL, H3: 20 µg/mL, H4: 40 µg/mL, and C1: control) and mercury chloride prepared in hydrogen-rich water (H5: 5 µg/mL, H6: 10 µg/mL, H7: 20 µg/mL, H8: 40 µg/mL, and C2: control) were injected into earthworms. The changes and reductions in some bands representing proteins, lipids, and polysaccharides (3280 cm−1, 2922 cm−1, 2855 cm−1, 1170 cm−1, and 1047 cm−1) showed that protective effects could occur in groups prepared with hydrogen-rich water. In the FTIR results, it was found that these bands in the H3 group were more affected and decreased by the influence of mercury on earthworms than the H7 group prepared with hydrogen. LC–MS analysis showed that the changes in some ions of the highest dose groups (H4 and H8) were different, and mercury caused oxidative DNA damage in earthworms. When the high-level application groups of mercury, i.e., H4 and H8 were compared with the controls, the ion exchange ([M + H] + ; m/z 283.1) representing the 8-Oxo-dG level in earthworms was higher in the H4 group than the H8 group. This reveals that HRW exhibited the potential ability to alleviate the toxic effects of mercury; however, a longer period of HRW treatment may be necessary to distinguish an obvious effect. The ATR-FTIR spectroscopy provided a rapid and precise method for monitoring the changes in biological tissues caused by a toxic compound at the molecular level.
Similar content being viewed by others
Introduction
Mercury (Hg) is a persistent and bioaccumulative toxic substance that is widely released into the environment (Schroeder and Munthe 1998). The toxic effects of mercury on aquatic animals and mammals are unquestioned (Gentès et al. 2015; Oliveira et al. 2017; Sun et al. 2019; Zhang et al. 2020a). However, the potential environmental risks of mercury in terrestrial systems remain largely uncertain (Tang et al. 2018). In some studies, the mortality increased, and the growth rate decreased when earthworms were exposed to mercury (Zhu et al., 2011; Da Silva et al. 2016), while death or growth inhibition was not observed in others (Lock and Janssen 2001; Mahbub et al. 2017). These differences in toxicity of mercury in earthworms may be due to exposure doses and durations. Buch et al. (2017) reported that mercury at 0–25 mg/kg caused a 10% inhibition effect on reproduction (EC10) of earthworms, while another study conducted at the same doses reported that mercury increased their reproduction (Abbasi and Soni 1983). However, this element is known at a wide scale by its toxicity at high doses and its amount in the ecosystem increases day by day (Programme 2013; Li et al. 2016). Bioaccumulation may be used as a bioindicator of mercury in the terrestrial environment (Dang et al. 2016; Le Roux et al. 2016). Toxicological screening of non-target (earthworm) has been considered as a vital endpoint in ecotoxicology study (Lankadurai et al. 2015; Ponsankar et al. 2020) with a key role of earthworm in the structure and nutrient value of soil (Vasantha-Srinivasan et al. 2016).
Hydrogen is the smallest and lightest molecule in nature with colorless and odorless properties (Alwazeer 2019). Due to its small size, molecular hydrogen can rapidly pass biomembranes to the cytoplasm, mitochondria, and nucleus to exert its protective effect (Ohta 2015; Zhai et al. 2017). Numerous studies showed that molecular hydrogen can protect various organs and tissues and has potential therapeutic properties for many diseases such as diabetes (Zhang et al. 2018), obesity (Nakao et al. 2010), and pancreatitis (Chen et al. 2010). Molecular hydrogen has been proven to possess a selective antioxidant activity against hydroxyl radicals (Ohsawa et al. 2007). Moreover, recent studies in animals, humans, and in vivo have shown that molecular hydrogen possesses therapeutic benefits in improving excessive inflammation and oxidative stress (Slezák et al. 2016; Yoneda et al. 2017; Jackson et al. 2018; Lu et al. 2019; Alwazeer et al. 2021). Hydrogen-rich water (HRW) is known to alleviate ischemia–reperfusion injury (impaired cardiac functions) in mice and decrease the oxidative stress level of myocardial tissue (Li et al. 2019). HRW showed also a protective effect on radiation-induced cognitive dysfunction thanks to its antioxidative and anti-inflammatory properties and its protection of neonatal neurons by regulating the BDNF-TrkB signaling pathway (Liu et al. 2019). In toxicological studies, the toxicity effect of a substance on the earthworm body is generally measured by either its lethal concentration or by its residue level in the tissue. Metabolic disturbance after exposing earthworm to a toxic substance could be measured by analyzing either the whole-worm homogenate or the coelomic fluid. However, these methods require homogenization and extraction procedures with extra steps for further molecular level analysis (Aja et al. 2014). Little information is available about the variation in the coelomic fluid of earthworms or its chemical complement in whole-body homogenates in response to toxic stress factors such as heavy metals.
Reactive oxygen species (ROSs) are highly reactive derivatives of oxygen molecules that form as a result of oxidative reactions in cells especially in mitochondria (Sarniak et al. 2016). ROSs play critical roles in a variety of cellular processes such as immune response and cell signaling when found at moderate levels (Magherini et al. 2019). However, when their level increases due to oxidative stress and exceeds the capacity of the antioxidant defense system, ROSs react with different cellular components such as proteins, lipids, deoxyribonucleotide triphosphates (dNTP), and DNA causing alteration of their structure and disruption of their physiological functions (Sies 1993; Thannickal and Fanburg 2000; Cooke et al. 2003). 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were typically used as critical biomarkers of oxidative stress-derived DNA damage (Ma et al. 2016; Pour Khavari and Haghdoost 2020; Wen et al. 2021). A significant increase of 8-oxo-dG in serum or urine was related to several diseases such as cancer, diabetes, and autoimmune diseases (Khavari et al. 2018; Thomas et al. 2018; Halczuk et al. 2020; Urbaniak et al. 2020). Techniques for 8-oxo-dG measurement include chromatographic approaches using GC–MS, GC–MS/MS, LC–MS/MS, and HPLC-ECD (Collins et al. 2000; Boysen et al. 2010). FTIR spectroscopy is a sensitive and highly reproducible physicochemical analytical technique that depends on measuring the wavelength and intensity of the absorption of IR radiation by the sample. The main advantages of FTIR over other techniques are that spectra can be obtained for a wide range of proteins, and direct correlations are possible between amide I band frequencies and secondary structure components (Aja et al. 2014). In another study, we proved that HRW could alleviate the nickel-induced toxic responses (inflammatory responses, oxidative stress, DNA damage) in the earthworm model (Köktürk et al. 2021). In the present study, we aim to screen the possible effects of hydrogen-rich water on mercury toxicity by interpreting biochemical changes in earthworms using ATR-FTIR and LC–MS analyses.
Materials and methods
Chemicals
Mercury(II) chloride (HgCl2) was purchased from Sigma. Stock solutions were prepared in both ultrapure water and hydrogen-rich water (Ultrapure water direct-Q®8 UV system) (HRW). Hydrogen-rich water was prepared using ultrapure water direct-Q®8 UV system (Millipore, USA). Liquid chromatography grade (LC) acetonitrile and methanol were purchased from Sigma-Aldrich. Analytical grade formic acid and ammonium formate were obtained from Sigma-Aldrich.
Preparation of hydrogen-rich water (HRW)
HRW was prepared according to Ryu et al. (Ryu et al. 2019) with some modifications. H2 gas was bubbled directly into mercury (II) chloride (HgCl2) solutions (5, 10, 20, and 40 μg/mL) for 10 min at 0.5 L/min with a hose equipped with a needle. The prepared solutions were then injected into earthworms within 5 min. The concentration of hydrogen in HRW samples and its stability were evaluated in preliminary experiments using the ORP electrode (Sensorex, USA). The levels of hydrogen were 1.6 ppm, and it did not change significantly during the assay period, i.e., 5 min (from HRW preparation moment to application one).
Animal study
Red California earthworms (Eisenia fetida) grown at the earthworm manure production unit at the College of Applied Sciences, Department of Organic Farming were used. Earthworms were kept at 20 ± 2 °C and 75% humidity and complete dark conditions until use. Healthy and adult soil worms (350–400 mg individual weight, adult earthworms) were selected for the experiment and kept under trial conditions in the laboratory 2 weeks before the experiment started.
The direct injection method was used by many researchers to investigate the toxic metabolism of chemicals in vivo in the coelomic cavity of earthworms (Nakatsugawa and Nelson 1972; Park et al. 2012; Yesudhason et al. 2018). Micro Fine Plus 0.5 mL (0.3 × 8.0 mm) needles were used. In this experiment, the earthworms were adapted to the test medium for 12 h on moist filter papers placed in a Petri dish with a vent hole. Before the direct injection, the earthworms were kept on ice for 10 min to cool down. The concentrations used in this study were chosen according to previous studies (Yesudhason et al. 2018). Twenty microliters of the HgCl2 solutions (5, 10, 20, and 40 µg/mL) was injected into the coelomic cavity of earthworms according to previous studies (Yesudhason et al. 2018; Kim et al. 2020). The application groups prepared with HRW were injected into the worms within 5 min after preparation so that the level of molecular hydrogen in water was not changed. In preliminary experiments, the hydrogen level in water was not significantly changed for few hours (data not shown). Different concentrations of mercury chloride (H1: 5 µg/mL, H2: 10 µg/mL, H3: 20 µg/mL, H4: 40 µg/mL, and C1: control) and mercury chloride prepared in hydrogen-rich water (H5: 5 µg/mL, H6: 10 µg/mL, H7: 20 µg/mL, H8: 40 µg/mL, and C2: control) were injected into earthworms. The experiments were designed with three replicates (n = 3) and 6 earthworms per group. During the experiment (48 h), the temperature was set at 20 °C and the humidity at 75%. Moist filter sheets were changed daily. When the analysis was terminated at 48 h, earthworms in each treatment group were washed with ultrapure water and placed in a Petri dish on a wet filter paper in the dark at 20 °C ± 2 °C for 12 h to remove their intestinal contents. The control and treated earthworms were directly freeze-dried using a lyophilizer (Christ Alpha 1–2 LD Plus, 2015, Germany), and samples were stored at − 80 °C until analysis.
ATR-FTIR spectroscopy
Six earthworms were taken from each test replicate, and after washing step with ultrapure water they were used for analysis. Details of FTIR-ATR techniques are available in previous studies (Rodriguez-Seijo et al. 2017; Paço et al. 2017; Rodríguez-Seijo et al. 2018). Fourier transform infrared-attenuated total reflectance (ATR-FTIR) analysis was performed using Agilent Cary 630 FTIR spectrometer. All spectra were obtained using 64 scans and a resolution of 4 cm−1 within the 4000–400 cm−1 range. Graphs of FTIR results were drawn using OriginPro 8 software. Each infrared spectrum was compared with the corresponding infrared spectra of all other different concentrations.
Since the ATR-FTIR spectrum of whole earthworm tissue is very complex and consists of various bands affiliated with the various functional groups in proteins, lipids, polysaccharides, and nucleic acids, the analyses were carried out in three different wavenumber regions, i.e., 4000–2800 cm−1 (C–H stretching region) and 1800–400 cm−1 (fingerprint region).
Table 1 shows that the (≠ 1) band 3280 cm−1 is mainly due to N–H vibrations in proteins, O–H stress vibrations in polysaccharides, and water. Since the water of the samples was completely removed, water has no contribution to this band, and only the contribution of proteins and polysaccharides could be taken into account (Çakmak et al. 2003; Cakmak et al. 2006; Cakmak-Arslan et al. 2020).
LC–ESI–MS/MS
Earthworm sample preparation
The sample preparation method for LC–ESI–MS/MS analysis was performed according to Wang et al. (Wang et al. 2014) and Griffith et al. (Griffith et al. 2019) with little modifications. Six lyophilized earthworms were taken from each group for LC–ESI–MS/MS analysis. Earthworm tissues of C2, H4, and H8 groups were prepared with methanolic solvent (50%). The samples were then vortexed for 2 min at 2000 rpm (Bioprep-24 Homogenizer) and 4 °C followed by centrifugation at 16,000 × g (Hettich Universal 320 R, Germany) at 4 °C for 20 min. The supernatant was then left in ice for 10 min until LC–MS analysis. All the solutions were filtrated before LC–ESI–MS/MS analysis using Captiva Premium Syringe Filter, polypropylene housing, nylon membrane (0.45 µm). The product and precursor ion of molecules were analyzed by LC–ESI–MS/MS.
Mass spectrometer and chromatography conditions
A 1260 Infinity II LC System model (Agilent, USA) High-Performance Liquid Chromatograph (HPLC) coupled with a Tandem Mass Spectrometer was used to carry out a qualitative evaluation ion of some molecules. The reversed-phase HPLC was equipped with a column oven (1260 TCC), binary pumps (1260 Bin Pump), and a degasser (1260 Degasser). The chromatographic conditions were optimized to achieve optimum separation of compounds and overcome the suppression effects. Thus, the chromatographic separation was performed on a reversed-phase Agilent Poroshell 120 EC-C18 model (100 mm × 3.0 mm, 2.7 μm) analytical column. The column temperature was set to 25 °C. The elution gradient was composed of eluent A (water + 5 mM ammonium formate) and eluent B (acetonitrile + 0.1% formic acid). The applied elution was 75% (A) –25% (B), and the solvent flow rate and injection volume were settled as 0.5 mL/min and 5 μL, respectively.
The mass spectrometric detection was carried out using an Agilent 6460 Triple Quad Mass Spectrometer System Model Tandem Mass Spectrometer equipped with an electrospray ionization (LC–ESI–MS/MS) source operating in both negative and positive ionization modes. LC–ESI–MS/MS data were acquired and processed by Agilent Mass Hunter Software. The MRM (Multiple Reaction Monitoring) method was optimized to selectively detect and quantify the phytochemical compounds based on the screening of specified precursor phytochemical-to-fragment ion transitions. The collision energies (CE) were optimized to generate optimal fragmentation and maximal transmission of the desired ions. The MS operating conditions were applied as follows: drying gas as nitrogen (N2) at a flow of 15 L/min; nebulizing gas as nitrogen at a flow of 11 L/min; and capillary (V) at 4000 V and gas temperature at 350 °C.
Statistical analysis
Analyses of variance (one-way ANOVA) with Tukey’s post hoc tests were used for multiple comparisons of FTIR band area values. One-way ANOVA has been performed with GraphPad Prism 9 Software. The degree of significance was denoted as **p < 0.01 and ***p < 0.001. FTIR band area values for the heat map chart were evaluated using the IBM SPPS v21 program, and the normality of the variables was evaluated by one-sample Kolmogorov–Smirnov and Shapiro–Wilk tests. Differences between groups were tested using one-way ANOVA for parametric data and the Mann–Whitney U test for non-parametric data. The values of p < 0.05 were considered significant in the evaluation of the data.
Results and discussion
ATR-FTIR study
The present study revealed that both ultrapure water-prepared HgCl2 (5, 10, 20, and 40 µg/mL) and hydrogen-rich ultrapure water-prepared HgCl2 concentrations (5, 10, 20, and 40 µg/mL) did not exhibit mortality effect against E. fetida. However, spectroscopic data of all cell structures can show structural and/or compositional changes occurring during cellular metabolic activity due to the effects of xenobiotics and environmental changes (stress factors) (Kamnev 2008; Mechirackal Balan et al. 2018). In this study, we evaluated the metabolic changes of earthworms exposed to HgCl2 stress using FTIR spectroscopic analysis. Besides, we examined how the different concentrations of HgCl2 prepared with hydrogen-rich water can affect the cellular metabolic activity compared to the control group, i.e., earthworms prepared with ultrapure water. Figures 1A, B and 3A–D show average ATR-FTIR spectra of C1, C2, H1, H2, H3, H4, H5, H6, H7, and H8-treated groups of earthworm. The main absorption bands were numbered in these figures, and the assignments of these bands were presented in Table 1 and Fig. 2.
Results show that there is no significant difference in some spectra (≠ 1 and ≠ 2) between the lowest concentrations of the treatment groups, i.e., H1 and H5 (Figs. 2 and 3A) (p˃0.05). However, when the H3 group is compared with the H7 application group, there is a significant difference in some spectra (≠ 1, ≠ 2, ≠ 3, ≠ 10, ≠ 11) (Figs. 2 and 3C) (p < 0.01, p < 0.001).
In the H2 and H6 application groups, we determined changes in the bands of 3278 cm−1 and 3279 cm−1, and in the H3 and H7 application groups, the bands of 3279 cm−1 and 3280 cm−1, respectively (Fig. 3B, C). When the 3280 cm−1 (≠ 1) band was evaluated for the H3 treatment group, it showed a more significant decrease than the H7 treatment group that was prepared with hydrogen-rich water (p < 0.01) (Figs. 2 and 4). Additionally, the low decrease in the polysaccharide band in the groups prepared with hydrogen-rich water could be related to the protective effect of molecular hydrogen. The decrease of this band in the H3 group indicates a decrease in the unsaturated lipid substances. The change in the bands and the shift in the frequency values are likely due to the change in lipid metabolism induced in the H3 and H7 groups. Thus, the mercury present in hydrogen-rich water may affect proteins and polysaccharides differently than other samples of middle dose (20 µg/mL). Different concentrations of mercury show an intense peak in 3420 cm−1 characteristics of an O–H stretch caused by intra-hydrogen and intermolecular bridges in the bands between 3100 and 3650 cm−1 in living beings, and a significant decrease in band density was observed in this bandgap at different concentrations of metals (Deguchi 2014; Rocha et al. 2020). This change of polysaccharide composition was supported by the change in the band of 1047 cm−1 (≠ 11) (Fig. 2) where the decrease in H2 (p < 0.01) and H3 groups (p < 0.001) compared with controls (C1 and C2) were more significant than H6 and H7 (Figs. 2 and 4). In addition, considering the same band, there was a significant decrease in the H3 group compared with the H7 group (p < 0.001) (Fig. 2).
When nickel and chromium metals were applied in Escherichia coli bacteria, the band determined as 1052 cm−1 in the control sample decreased to 1049 cm−1 and 1047 cm−1 in the nickel and chromium application groups, respectively (Gupta and Karthikeyan 2016). This change of polysaccharides could be due to the change of the peptidoglycan surface density of the cell wall to better adapt to their environment under heavy metal stress (Vollmer et al. 2008). Polysaccharides inhibit fatigue and bone loss activities as well as have a protective effect against oxidative stress and injuries (Yuan et al. 2019).
The CH2 antisymmetric stretching at 2919 cm−1 and the CH2 symmetric stretching at 2851 cm−1 is associated with saturated lipid concentration of membranes. The concentration of saturated lipids is important for the detection of membrane fluidity levels (Markowicz et al., 2010; Kardas et al. 2014). Antisymmetric and symmetrical methyl (vasCH3, vsCH3) and methylene (vasCH2, vsCH2) groups in spectral 3000–2850 cm−1 regions have tensile vibrations of lipid and phospholipids, and 2850 cm−1 IR absorption bands are used to monitor lipid changes (Mendelsohn and Moore 1998; Lewis and McElhaney 2007, 2013). In the present study, the band of 2855 cm−1 (≠ 3) (Fig. 2) shows a more significant decrease in H3 treatment groups compared with the H7 group (p < 0.001) (Figs. 2 and 4). In the groups without hydrogen-rich water application, changes in the 2855 cm−1 band may lead to changes in the permeability of the membranes, the higher order of lipophilic carbon–carbon chains, and a significant increase in saturated lipid concentrations (Kepenek et al. 2019). The decrease in saturated lipid concentration may result from decreased lipid biosynthesis or degradation through lipid peroxidation (Simsek Ozek et al. 2014). However, the absolute frequency of CH stretch bands can no longer be used to predict the degree of relative lipid impairment, and a low value of these parameters also indicates the presence of a long chain of fatty acids (Staniszewska et al. 2014). Besides, the density of CH stretch bands increases with advancing age and the development of diseases (Anastassopoulou et al. 2018).
The molecular changes in organisms exposed to stressors could be used to understand the responses to stress agents, and the peak at ≈1540 cm−1 corresponds to amide II structures (G. Muthukaruppan 2015; Rodriguez-Seijo et al. 2017). Table 1 (≠ 6) shows the FTIR-ATR spectra (1536 cm−1) of the earthworms after exposure to different concentrations of HgCl2 and controls. The band 1536 cm−1 in the H3 groups prepared without hydrogen-rich water shows a significant difference compared to the controls (C1 and C2) and H7 (p˂0.05) (Fig. 4). Similarly, the effect of xenobiotics in mice and the density and frequency of amide II bands (1453 cm−1 and 1525 cm−1) was decreased due to asymmetric methyl deformation and stretching of the C = N, C = C groups compared to the control (Ashtarinezhad et al. 2014, 2015).
Comparing to the control groups (C1 and C2), there was no significant difference in H7 of the hydrogen-rich application groups in the 1230 cm−1 band (≠ 9), while a significant decrease was observed in the H3 group (p < 0.05) (Fig. 4). The intensity and frequency of the bands around 1256 cm−1 and 1219 cm−1 in treated tissue were reduced and shifted compared to untreated sample tissue, mainly owing to PO2− asymmetric (phosphate I; 1256 cm−1) and PO2− asymmetric vibrations of nucleic acids when it is highly hydrogen-bonded asymmetric hydrogen-bonded phosphate stretching mode (1219 cm−1) (Ashtarinezhad et al. 2014). The phosphate band at 1230 cm−1 can originate from the phosphate backbone of the nucleic acid (Boydston-White et al. 2006). Higher phosphate absorption in the induced fibroblast indicates low compact nucleic acid materials potentially associated with increased nucleic acid synthesis activity. For induced fibroblasts, higher absorption at 2950 cm−1 and 1230 cm−1 wavenumbers has been reported to indicate an accelerated cell growth and division (Kumar et al. 2014). It has been proven that highly compact DNA has a reduced absorbency rather than the presence of fewer DNA molecules (Whelan et al. 2013).
LC–ESI–MS/MS study
To support ATR-FTIR results, we analyzed the ion changes in earthworm tissues of the highest concentration application groups, i.e., H4 and H8 by LC–ESI–MS/MS analysis using Agilent 6460 Triple Quadrupole Mass Spectrometer with Mass Detection for a targeted method. The highest response to molecular ions was obtained. C2, H4, and H8 application groups were infused and analyzed by LC–ESI–MS/MS. When the system was operated under full scan conditions in the negative and positive ion modes, data were collected in the range of m/z 100–1300 with a scan time of 0.5 s. The ESI–MS/MS system was used with nitrogen as the collision gas and fragmentor voltage of 135 V. The results showed that ions found in the control (C2) and H8 application groups ([M + H] + ; m/z 542.3), ([M + H] + ; m/z 706.6), and ([M + H] + ; m/z 798.6) were not detected in the H4 treatment groups (Fig. 5). Differently, the ions ([M − H] + ; m / z 533.3) and ([M − H] + ; m / z 605.4) present in H4 and control (C2) were not found in the H8 application (Fig. 5). LC–MS results show that when the H4 application group was compared with control (C2) and H8, some ions ([M + H] + ; m/z 276.1), ([M + H] + ; m/z 325.2), ([M + H] + ; m/z 283.1), ([M + H] + ; m/z 233.1), and ([M + H] + ; m/z 140.1) were increased, while other ions ([M + H] + ; m/z 184.1), ([M + H] + ; m/z 132), and ([M + H] + ; m/z 138.1) were decreased (Fig. 5).
Mostly similar ions were detected in H4 and C2 groups in the H8 treatment group. Whereas there were ions in the H8 application group and control ([M + H] + ; m/z 542.3, m/z 706.6, and m/z 796.8), these ions were not shown in the H4 application group; however, ([M + H] + m/z 298) ion was shown (Fig. 5). Differently, the negative ions ([M − H] − ; m/z 233, m/z 533.3, and m/z 605.4) were also present in H4 and C2 but not detected in the H8 application (Fig. 6).
Different peak intensities of similar ions were observed in the H8 treatment group, H4, and C2 group. As seen in Fig. 5, the positive ions in the H4 treatment group ([M + H] + ; m/z 276.1, m/z 325.2, m/z 140.1, m/z 283.1, m/z 118.1, and m/z 233.1) were detected at higher levels than the H8 treatment group and the C2. Again, from negative ions ([M − H] − ; m/z 275.2 and m/z 307.2), ions were higher in group H4 (Fig. 6).
From the DNA bases, deoxyguanosine (dG) is the most easily oxidized DNA base and forms 8-oxo-2′-deoxyguanosine (8-oxo-dG) which has a very low redox potential due to its 8-position (Boiteux and Radicella 1999). The formed 8-oxo-dG can pair with adenine and lead to G:C → T:A transversion mutations if not repaired before replication (Hsu et al. 2004). The interaction of reactive oxygen species with DNA causes various modifications in 8-oxo-7,8-dihydro-2′- deoxyguanosine (8-oxodG) which has been extensively studied as a biomarker of oxidative stress (Lam et al. 2012). Oxidative DNA lesion measurements, which cause age-related macular degeneration (AMD), were measured by the LC–MS method in retinal pigment epithelial cells, and when the oxidative DNA lesion biomarker such as 8-oxo-2′-deoxyguanosine (8-oxo-dG) was examined, the ion exchange was m/z 284.0 and m/z 287.0 ions change, and the 8-oxo-dG levels were found to be increased (Ma et al. 2016). In this study, when the high-level application groups of mercury, i.e., H4 and H8 were compared with the controls, the ion exchange ([M + H] + ; m/z 283.1) representing the 8-oxo-dG level earthworms was higher in the H4 group than the H8 group (Figs. 5 and 7). However, we found that the same ion was closer to each other in C2 compared with the H8 application group (Figs. 5 and 7). Although hydrogen-rich water (HRW) has been used to prevent various oxidative stress-related diseases, the underlying mechanisms remain unclear. It has been determined that HRW alleviates mercury toxicity in plants by reducing Hg accumulation, prevents the formation of oxidative stress, and re-establishes redox homeostasis (Kosikowska et al. 2010; Cui et al. 2014). However, no studies are revealing the mitigating effect of HRW on mercury or any heavy metal toxicity in vertebrates or invertebrates. Otherwise, it has been reported that HRW consumption reduces the total ROS level and decreases the frequency of DNA damage due to ROS formation (Ohsawa et al. 2007; Suzuki et al. 2017; Zhang et al. 2017). It was also found that H2 restored the balance of the redox state and suppressed oxidative stress damage by decreasing ROS and MDA levels while increasing CAT, GSH, and SOD activity (Lu et al. 2020). Similarly, according to the findings of our study, it can be assumed that changes in the level of 8-oxo-dG, that is a marker of oxidative stress-induced DNA damage were decreased in the H8 earthworm group prepared with HRW due to the decrease of the mercury accumulation in the presence of hydrogen-rich water, and accordingly, the DNA damage was reduced. Membrane phospholipids consist of abundant polyunsaturated fatty acids. Thus, lipid peroxidation can occur in both nuclear and mitochondrial membranes. Especially in the inner mitochondrial membrane, the mitochondrial electron transfer system can easily generate superoxide anions and peroxidize the membranous lipid (Ježek and Hlavatá 2005; Henderson et al. 2009). The formed lipid peroxides are thought to easily attack mitochondrial DNA because they are located close to the inner membrane, and it produces 8-oxo-dG by induction of lipid peroxidation in mitochondria (Hruszkewycz and Bergtold 1990). It has been reported that DNA produces 8-oxo-dG when mixed with peroxidizing lipids (Park and Floyd 1992). In this case, we can assume by the support of the above-cited reports that in mercury solutions prepared with hydrogen-rich water, there is less lipid peroxidation and less formed 8-oxo-dG. Besides, when the bands (≠ 2 and ≠ 3) related to lipid materials were evaluated in FTIR results (Figs. 2 and 4), the H8 application did not differ from C2 and H4 application groups. But the decrease (1454 cm−1) in earthworm tissues of the H8 group was less than the H4 group when the band of 1454 cm−1 (≠ 7) (Fig. 4) was compared with the C1 and C2 control groups. It has been reported that the peak at 1457 cm−1 formed from the toxicity of xenobiotics in tissues showed weak DNA and RNA peaks due to vibration of methyl and methylene protein and lipid groups (Dhakshinamoorthy et al. 2017). In this case, it was thought that DNA and RNA might be less affected in earthworms injected with mercury solutions prepared with hydrogen-rich water. Molecular hydrogen has been proven to exhibit a mild but effective antioxidant activity by rapid diffusion into tissues and cells (Ohsawa et al. 2007). Although molecular hydrogen has been characterized as an effective antioxidant, it shows non-side effects, and it is mild enough to not disrupt metabolic and redox reactions nor affect the mild ROSs that play a role in the cell signaling system (Salganik 2001; Sauer et al. 2001; Liu et al. 2005). In cells stimulated with xenobiotics, mitochondrial membrane permeability can increase, and intense mitochondrial reactive oxygen species (mtROS) can be produced with potential oxidation of mitochondrial DNA (mtDNA) (Shimada et al. 2012; Guo et al. 2019; Zhang et al. 2020b). Removal of mtROS by molecular hydrogen has been reported to reduce the formation of oxidized mitochondrial DNA (Ren et al. 2016). Similarly, studies showed that molecular hydrogen can prevent the reduction of mitochondrial membrane potential, protect mitochondria from •OH radicals, prevent the decrease in the cellular level of ATP synthesized in mitochondria, and protect mitochondria and nuclear DNA (Ohsawa et al. 2007; Ohta 2011; Xin et al. 2014). Molecular hydrogen is thought to function both as a radical scavenger against oxidative stress in cells and as a mitohormetic effector in moderate mitochondrial stress (Murakami et al. 2017). In the sugar-phosphate DNA backbone region, 1300–800 cm−1, more than six bands come into focus: asymmetric and symmetric PO2 stretching mode at 1235 and 1089 cm−1, respectively, sugar-phosphate stretching band at 1070 cm−1, deoxyribose stretching mode at 966 cm−1, and A and B form markers situated at about 860 cm−1 and 835 cm−1, respectively (Tsuboi, 1970). In addition, the absorption bands 1150 cm−1 and 1020–1025 cm−1 can be assigned to the C–O bond of glycogen and other carbohydrates and are significantly overlapped by DNA (Heidarpoor Saremi et al. 2021). Some ions, which are important in the DNA structure, affect them and cause vibration and rotational movements of the above-mentioned bonds. In our study, it was determined that there was a decrease in the 1170−1 and 1047 cm−1 bands in the H3 group compared to the H7 group in the range of 1300–800 cm−1. In this case, we can assume that the changes in the bonds affect less the DNA structure of the H7 group prepared with HRW and that HRW could alleviate the negative effects of mercury on DNA. The above-cited studies explain that the ion exchange and FTIR spectra band area value changes can give information about DNA, lipid, and protein in both FTIR and LC–ESI–MS/MS results, which show that hydrogen-rich water exhibits protective effects on earthworms in the case of mercury toxication in the present study.
The reason behind the absence of an obvious effect of HRW on the detoxification of mercury in earthworms could be attributed to the short application period of HRW and the high toxicity of mercury. In our recently published article, we have found that HRW could alleviate the effects of nickel toxicity in an earthworm model (Köktürk et al. 2021). In the previous study, a 2-week treatment with HRW was performed; however, in the present study, the HRW treatment was carried out only at the injection time of mercury. This shows that HRW would have exhibited potent and clear effects towards a heavy metal toxification if the HRW treatment time was longer like the 2-week HRW treatment performed in earthworms for the detoxification of nickel (Köktürk et al. 2021). To verify this hypothesis, it is important to extend the HRW treatment to cover a long period and perform additional analyses like immunohistochemical and immunofluorescence measurement.
Molecular hydrogen is known for its apparent inertness towards metals and nometals due to its very high dissociation energy, and it can reduce the oxides of most metals and many metallic salts to the metals only at high temperatures and pressures (William Lee Jolly 2020). As the physiological conditions of worms and assays (temperature and pressure) were not appropriate to this reaction between mercury chloride and molecular hydrogen, we assumed the absence of changes in the levels of mercury chloride during assays. However, the determination of mercury in water, HRW, and worm tissue samples forms a limitation of the study and should be also the topic of further study. The exact mechanism of the hydrogen effect on mercury and its chemical form change should form also an interesting issue of further study. Moreover, the study of the alleviation effects of HRW on DNA by exploring some additional markers in the DNA repair mechanism should also form another research subject.
Conclusion
The present work evaluated the use of hydrogen-rich water in terms of the detoxification purpose of mercury in the earthworm model, and it could show that hydrogen-rich water could mitigate the effects of toxic substances such as mercury. This study revealed that although hydrogen-rich water did not completely alleviate mercury toxicity, the results of ATR-FTIR differed from that of the normal water, i.e., without molecular hydrogen in terms of some ions and molecules. For the first time, biological monitoring of mercury-related oxidative stress could be achieved in earthworm tissues, and the formation of 8-oxodG was rapidly identified using LC–ESI–MS/MS method. These results are important for investigating innovative, friend-to-environment, and cost-effective methods for decontaminating environments contaminated with heavy metals. Additionally, this study provides an alternative approach to investigating the protective effects of hydrogen-rich water in ecotoxicological situations. These results provide a green method solution for the treatment of the problem of heavy metal toxification phenomenon in industrial regions.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Abbasi SA, Soni R (1983) Stress-induced enhancement of reproduction in earthworm octochaetus pattoni exposed to chromium (vi) and mercury (ii)— implications in environmental management. Int J Environ Stud 22:43–47. https://doi.org/10.1080/00207238308710100
Aja M, Jaya M, Vijayakumaran Nair K, Hubert Joe I (2014) FT-IR spectroscopy as a sentinel technology in earthworm toxicology. Spectrochim Acta - Part A Mol Biomol Spectrosc 120:534–541. https://doi.org/10.1016/j.saa.2013.12.004
Alwazeer D (2019) Reducing atmosphere packaging technique for extending the shelf-life of food products. J Inst Sci Technol 9:2117–2123. https://doi.org/10.21597/jist.539744
Alwazeer D, Liu FF-C , Wu XY, LeBaron WT (2021) Combating oxidative stress and inflammation in COVID-19 by molecular hydrogen therapy: mechanisms and perspectives. Oxid Med Cell Longev. https://doi.org/10.1155/2021/5513868
Anastassopoulou J, Kyriakidou M, Kyriazis S et al (2018) Oxidative stress in ageing and disease development studied by FT-IR spectroscopy. Mech Ageing Dev 172:107–114. https://doi.org/10.1016/j.mad.2017.11.009
Ashtarinezhad A, Panahyab A, Mohamadzadehasl B (et al2015) FTIR microspectroscopy reveals chemical changes in mice fetus following phenobarbital administration. Iran J Pharm Res 14:121–130. https://doi.org/10.22037/ijpr.2015.1721
Ashtarinezhad A, Shirazi FH, Vatanpour H et al (2014) FTIR-microspectroscopy detection of metronidazole teratogenic effects on mice fetus. Iran J Pharm Res 13:101–111. https://doi.org/10.22037/ijpr.2014.1464
Boiteux S, Radicella JP (1999) Base excision repair of 8-hydroxyguanine protects DNA from endogenous oxidative stress. Biochimie 81:59–67. https://doi.org/10.1016/S0300-9084(99)80039-X
Boydston-White S, Romeo M, Chernenko T et al (2006) Cell-cycle-dependent variations in FTIR micro-spectra of single proliferating HeLa cells: principal component and artificial neural network analysis. Biochim Biophys Acta - Biomembr 1758:908–914. https://doi.org/10.1016/j.bbamem.2006.04.018
Boysen G, Collins LB, Liao S et al (2010) Analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine by ultra high pressure liquid chromatography-heat assisted electrospray ionization-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 878:375–380. https://doi.org/10.1016/j.jchromb.2009.12.004
Buch AC, Brown GG, Correia MEF et al (2017) Ecotoxicology of mercury in tropical forest soils: Impact on earthworms. Sci Total Environ 589:222–231. https://doi.org/10.1016/j.scitotenv.2017.02.150
Cakmak-Arslan G, Haksoy H, Goc-Rasgele P, Kekecoglu M (2020) Determination of the dose-dependent toxic effects of mad honey on mouse liver using ATR-FTIR spectroscopy. Spectrochim Acta - Part A Mol Biomol Spectrosc 228:117719. https://doi.org/10.1016/j.saa.2019.117719
Cakmak G, Togan I, Severcan F (2006) 17β-Estradiol induced compositional, structural and functional changes in rainbow trout liver, revealed by FT-IR spectroscopy: a comparative study with nonylphenol. Aquat Toxicol 77:53–63. https://doi.org/10.1016/j.aquatox.2005.10.015
Çakmak G, Togan I, Uǧuz C, Severcan F (2003) FT-IR spectroscopic analysis of rainbow trout liver exposed to nonylphenol. Appl Spectrosc 57:835–841. https://doi.org/10.1366/000370203322102933
Chen H, Sun YP, Li Y et al (2010) Hydrogen-rich saline ameliorates the severity of l-arginine-induced acute pancreatitis in rats. Biochem Biophys Res Commun 393:308–313. https://doi.org/10.1016/j.bbrc.2010.02.005
Collins A, Brown J, Bogdanov M et al (2000) Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage. Free Radic Res 32:333–341. https://doi.org/10.1080/10715760000300331
Cooke MS, Evans MD, Dizdaroglu M, Lunec J (2003) Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J 17:1195–1214. https://doi.org/10.1096/fj.02-0752rev
Cui W, Fang P, Zhu K et al (2014) Hydrogen-rich water confers plant tolerance to mercury toxicity in alfalfa seedlings. Ecotoxicol Environ Saf 105:103–111. https://doi.org/10.1016/j.ecoenv.2014.04.009
Da Silva E, Nahmani J, Lapied E et al (2016) Toxicity of mercury to the earthworm Pontoscolex corethrurus in a tropical soil of French Guiana. Appl Soil Ecol 104:79–84. https://doi.org/10.1016/j.apsoil.2015.11.018
Dang F, Zhao J, Zhou D (2016) Uptake dynamics of inorganic mercury and methylmercury by the earthworm Pheretima guillemi. Chemosphere 144:2121–2126. https://doi.org/10.1016/j.chemosphere.2015.10.111
Deguchi TGF (2014) Estudo do equilíbrio químico de compostos modelo de taninos com íons metálicos para o tratamento de efluentes industriais. 113
Dhakshinamoorthy V, Manickam V, Perumal E (2017) Neurobehavioural toxicity of iron oxide nanoparticles in mice. Neurotox Res 32:187–203. https://doi.org/10.1007/s12640-017-9721-1
Muthukaruppan G (2015) Heavy metal induced biomolecule and genotoxic changes in earthworm Eisenia fetida | Invertebrate Survival Journal. Invertebr Surviv J 12:237–245
Gentès S, Maury-Brachet R, Feng C et al (2015) Specific effects of dietary methylmercury and inorganic mercury in zebrafish (Danio rerio) determined by genetic, histological, and metallothionein responses. Environ Sci Technol 49:14560–14569. https://doi.org/10.1021/acs.est.5b03586
Griffith CM, Thai AC, Larive CK (2019) Metabolite biomarkers of chlorothalonil exposure in earthworms, coelomic fluid, and coelomocytes. Sci Total Environ 681:435–443. https://doi.org/10.1016/j.scitotenv.2019.04.312
Guo H, Liu H, Jian Z et al (2019) Nickel induces inflammatory activation via NF-kB, MAPKs, IRF3 and NLRP3 inflammasome signaling pathways in macrophages. Aging (Albany NY) 11:11659–11672. https://doi.org/10.18632/aging.102570
Gupta AD, Karthikeyan S (2016) Individual and combined toxic effect of nickel and chromium on biochemical constituents in E. coli using FTIR spectroscopy and Principle component analysis. Ecotoxicol Environ Saf 130:289–294. https://doi.org/10.1016/j.ecoenv.2016.04.025
Halczuk KM, Boguszewska K, Urbaniak SK, et al (2020) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) as a cause of autoimmune thyroid diseases (AITD) during pregnancy?
Heidarpoor Saremi L, Dadashi Noshahr K, Ebrahimi A, et al (2021) Multi-stage screening to predict the specific anticancer activity of Ni(II) mixed-ligand complex on gastric cancer cells; biological activity, FTIR spectrum, DNA binding behavior and simulation studies Spectrochimica Acta Part A: Molecular and Biomolecula. https://doi.org/10.1016/j.saa.2020.119377
Henderson JR, Swalwell H, Boulton S et al (2009) Direct, real-time monitoring of superoxide generation in isolated mitochondria. Free Radic Res 43:796–802. https://doi.org/10.1080/10715760903062895
Hruszkewycz AM, Bergtold DS (1990) The 8-hydroxyguanine content of isolated mitochondria increases with lipid peroxidation. Mutat Res Lett 244:123–128. https://doi.org/10.1016/0165-7992(90)90060-W
Hsu GW, Ober M, Carell T, Beese LS (2004) Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature 431:217–221. https://doi.org/10.1038/nature02908
Jackson K, Dressler N, Ben-Shushan RS et al (2018) Effects of alkaline-electrolyzed and hydrogen-rich water, in a high-fat-diet nonalcoholic fatty liver disease mouse model. World J Gastroenterol 24:5095–5108. https://doi.org/10.3748/wjg.v24.i45.5095
Ježek P, Hlavatá L (2005) Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int J Biochem Cell Biol 37:2478–2503
Kamnev AA (2008) FTIR spectroscopic studies of bacterial cellular responses to environmental factors, plant-bacterial interactions and signalling. Spectroscopy 22:83–95
Kardas M, Gozen AG, Severcan F (2014) FTIR spectroscopy offers hints towards widespread molecular changes in cobalt-acclimated freshwater bacteria. Aquat Toxicol 155:15–23. https://doi.org/10.1016/j.aquatox.2014.05.027
Kepenek ES, Gozen AG, Severcan F (2019) Molecular characterization of acutely and gradually heavy metal acclimated aquatic bacteria by FTIR spectroscopy. J Biophotonics 12(5):e201800301. https://doi.org/10.1002/jbio.201800301
Khavari AP, Liu Y, He E, et al (2018) Serum 8-oxo-dG as a predictor of sensitivity and outcome of radiotherapy and chemotherapy of upper gastrointestinal tumours. Oxid Med Cell Longev 2018https://doi.org/10.1155/2018/4153574
Kim S, Jeon D, Lee JY et al (2020) Upregulation of cellulase activity and mRNA levels by bacterial challenge in the earthworm Eisenia andrei, supporting the involvement of cellulases in innate immunity. Biochem Biophys Res Commun 521:15–18. https://doi.org/10.1016/j.bbrc.2019.09.134
Köktürk M, Yıldırım S, Eser G, et al (2021) Hydrogen-rich water alleviates the nickel-induced toxic responses (inflammatory responses, oxidative stress, DNA damage) and ameliorates cocoon production in earthworm. Biol Trace Elem Res 1–11
Kosikowska U, Smolarz HD, Malm A (2010) Antimicrobial activity and total content of polyphenols of Rheum L. species growing in Poland. Cent Eur J Biol 5:814–820. https://doi.org/10.2478/s11535-010-0067-4
Kumar S, Shabi TS, Goormaghtigh E (2014) A FTIR imaging characterization of fibroblasts stimulated by various breast cancer cell lines. PLoS ONE 9(11):e111137. https://doi.org/10.1371/journal.pone.0111137
Lam PMW, Mistry V, Marczylo TH et al (2012) Rapid measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in human biological matrices using ultra-high-performance liquid chromatography-tandem mass spectrometry. Free Radic Biol Med 52:2057–2063. https://doi.org/10.1016/j.freeradbiomed.2012.03.004
Lankadurai BP, Nagato EG, Simpson AJ, Simpson MJ (2015) Analysis of Eisenia fetida earthworm responses to sub-lethal C60 nanoparticle exposure using 1H-NMR based metabolomics. Ecotoxicol Environ Saf 120:48–58. https://doi.org/10.1016/j.ecoenv.2015.05.020
Le Roux S, Baker P, Crouch A (2016) Bioaccumulation of total mercury in the earthworm Eisenia andrei. Springerplus 5:681. https://doi.org/10.1186/s40064-016-2282-6
Lewis RNAH, McElhaney RN (2007) Fourier Transform infrared spectroscopy in the study of Lipid phase transitions in model and biological membranes. in: Methods in molecular biology (Clifton, N.J.). pp 207–226
Lewis RNAH, McElhaney RN (2013) Membrane lipid phase transitions and phase organization studied by Fourier transform infrared spectroscopy. Biochim Biophys Acta - Biomembr 1828:2347–2358
Li L, Liu T, Liu L et al (2019) Effect of hydrogen-rich water on the Nrf2/ARE signaling pathway in rats with myocardial ischemia-reperfusion injury. J Bioenerg Biomembr 51:393–402. https://doi.org/10.1007/s10863-019-09814-7
Li Y, Ma C, Zhu C et al (2016) Historical anthropogenic contributions to mercury accumulation recorded by a peat core from Dajiuhu montane mire, central China. Environ Pollut 216:332–339. https://doi.org/10.1016/j.envpol.2016.05.083
Liu H, Colavitti R, Rovira II, Finkel T (2005) Redox-dependent transcriptional regulation. Circ Res 97:967–974. https://doi.org/10.1161/01.RES.0000188210.72062.10
Liu M, Yuan H, Yin J et al (2019) Effect of hydrogen-rich water on radiation-induced cognitive dysfunction in rats. Radiat Res 193:16. https://doi.org/10.1667/RR15464.1
Lock K, Janssen C (2001) Ecotoxicity of mercury to Eisenia fetida, Enchytraeus albidus and Folsomia candida. Biol Fertil Soils 34:219–221. https://doi.org/10.1007/s003740100392
Lu R, Liu Y, Wang D (2019) Protective effect of hydrogen-rich water on oxidative stress cell model and the impact of the phosphatidylinositol 3 kinase/protein kinase B pathway. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue 31:762–767. https://doi.org/10.3760/cma.j.issn.2095-4352.2019.06.020
Lu Y, Li CF, Ping NN et al (2020) Hydrogen-rich water alleviates cyclosporine A-induced nephrotoxicity via the Keap1/Nrf2 signaling pathway. J Biochem Mol Toxicol 34:1–9. https://doi.org/10.1002/jbt.22467
Ma B, Jing M, Villalta PW et al (2016) Simultaneous determination of 8-oxo-2-deoxyguanosine and 8-oxo-2-deoxyadenosine in human retinal DNA by liquid chromatography nanoelectrospray-tandem mass spectrometry. Sci Rep 23(1):151–60. https://doi.org/10.1038/srep22375
Magherini F, Fiaschi T, Marzocchini R et al (2019) Oxidative stress in exercise training: the involvement of inflammation and peripheral signals. Free Radic Res 53:1155–1165
Mahbub KR, Krishnan K, Naidu R et al (2017) Mercury toxicity to terrestrial biota. Ecol Indic 74:451–462
Markowicz A, Płociniczak T, Piotrowska-Seget Z (2010) Response of bacteria to heavy metals measured as changes in FAME profiles. Polish J Environ Stud 19:957–965
Mechirackal Balan B, Shini S, Krishnan KP, Mohan M (2018) Mercury tolerance and biosorption in bacteria isolated from Ny-Ålesund, Svalbard, Arctic. J Basic Microbiol 58:286–295. https://doi.org/10.1002/jobm.201700496
Mendelsohn R, Moore DJ (1998) Vibrational spectroscopic studies of lipid domains in biomembranes and model systems. Chem Phys Lipids 96:141–157. https://doi.org/10.1016/S0009-3084(98)00085-1
Murakami Y, Ito M, Ohsawa I (2017) Molecular hydrogen protects against oxidative stress-induced SH-SY5Y neuroblastoma cell death through the process of mitohormesis. PLoS ONE 12:e0176992–e0176992. https://doi.org/10.1371/journal.pone.0176992
Nakao A, Toyoda Y, Sharma P et al (2010) Effectiveness of hydrogen rich water on antioxidant status of subjects with potential metabolic syndrome—an open label pilot study. J Clin Biochem Nutr 46:140–149
Nakatsugawa, T. and Nelson PA (1972) Studies of insecticide detoxication in invertebrates; an enzymological approach to the problem of biological magnification. Environ Toxicol Pestic 501–524
Ohsawa I, Ishikawa M, Takahashi K et al (2007) Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 13:688
Ohta S (2015) Molecular hydrogen as a novel antioxidant: overview of the advantages of hydrogen for medical applications. In: Methods in enzymology. Elsevier, pp 289–317
Ohta S (2011) Recent progress toward hydrogen medicine: potential of molecular hydrogen for preventive and therapeutic applications. Curr Pharm Des 17:2241–2252
Oliveira VA, Favero G, Stacchiotti A et al (2017) Acute mercury exposition of virgin, pregnant, and lactating rats: histopathological kidney and liver evaluations. Environ Toxicol 32:1500–1512. https://doi.org/10.1002/tox.22370
Paço A, Duarte K, da Costa JP et al (2017) Biodegradation of polyethylene microplastics by the marine fungus Zalerion maritimum. Sci Total Environ 586:10–15. https://doi.org/10.1016/j.scitotenv.2017.02.017
Park B-S, Yoo J-H, Kim J-H et al (2012) Biotransformation of endosulfan by the tiger worm, Eisenia fetida. J Agric Chem Environ 01:20–27. https://doi.org/10.4236/jacen.2012.11004
Park JW, Floyd RA (1992) Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA. Free Radic Biol Med 12:245–250. https://doi.org/10.1016/0891-5849(92)90111-S
Ponsankar A, Sahayaraj K, Senthil-Nathan S et al (2020) Toxicity and developmental effect of cucurbitacin E from Citrullus colocynthis L. (Cucurbitales: Cucurbitaceae) against Spodoptera litura Fab. and a non-target earthworm Eisenia fetida Savigny. Environ Sci Pollut Res 27:23390–23401. https://doi.org/10.1007/s11356-019-04438-1
Pour Khavari A, Haghdoost S (2020) Effects of tomato juice intake on salivary 8-oxo-dG levels as oxidative stress biomarker after extensive physical exercise. Oxid Med Cell Longev 2020:8948723. https://doi.org/10.1155/2020/8948723
Programme UNE (2013) United Nations Environment Programme Global Mercury Assessment 2013 Sources, Emissions, Releases and Environmental Transport
Ren JD, Wu XB, Jiang R et al (2016) Molecular hydrogen inhibits lipopolysaccharide-triggered NLRP3 inflammasome activation in macrophages by targeting the mitochondrial reactive oxygen species. Biochim Biophys Acta - Mol Cell Res 1863:50–55. https://doi.org/10.1016/j.bbamcr.2015.10.012
Rocha JE, Guedes TTAM, Bezerra CF et al (2020) FTIR analysis of pyrogallol and phytotoxicity-reductive effect against mercury chloride. Environ Geochem Health 43(6):2433–2442. https://doi.org/10.1007/s10653-020-00607-1
Rodríguez-Seijo A, da Costa JP, Rocha-Santos T et al (2018) Oxidative stress, energy metabolism and molecular responses of earthworms (Eisenia fetida) exposed to low-density polyethylene microplastics. Environ Sci Pollut Res 25:33599–33610. https://doi.org/10.1007/s11356-018-3317-z
Rodriguez-Seijo A, Lourenço J, Rocha-Santos TAP et al (2017) Histopathological and molecular effects of microplastics in Eisenia andrei Bouché. Environ Pollut 220:495–503. https://doi.org/10.1016/j.envpol.2016.09.092
Ryu J, Kim MJ, Lee JH (2019) Extraction of green tea phenolics using water bubbled with gases. J Food Sci 84:1308–1314. https://doi.org/10.1111/1750-3841.14606
Salganik RI (2001) The benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population. J Am Coll Nutr 464S-472S
Sarniak A, Lipińska J, Tytman K, Lipińska S (2016) Endogenous mechanisms of reactive oxygen species (ROS) generation. Postepy Hig Med Dosw 70:1150–1165. https://doi.org/10.5604/17322693.1224259
Sauer H, Wartenberg M, Hescheler J (2001) Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 11:173–186. https://doi.org/10.1159/000047804
Schroeder WH, Munthe J (1998) Atmospheric mercury—an overview. In: Atmospheric environment. Pergamon, pp 809–822
Shimada K, Crother TR, Karlin J et al (2012) Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 36:401–414. https://doi.org/10.1016/j.immuni.2012.01.009
Sies H (1993) Strategies of Antioxidant defense: relations to oxidative stress. Signal Mech — from Transcr Factors to Oxidative Stress 215:213–219. https://doi.org/10.1007/978-3-642-79675-3_15
Simsek Ozek N, Bal IB, Sara Y et al (2014) Structural and functional characterization of simvastatin-induced myotoxicity in different skeletal muscles. Biochim Biophys Acta - Gen Subj 1840:406–415. https://doi.org/10.1016/j.bbagen.2013.09.010
Slezák J, Kura B, Frimmel K et al (2016) Preventive and therapeutic application of molecular hydrogen in situations with excessive production of free radicals. Physiol Res 65:S11–S28
Staniszewska E, Malek K, Baranska M (2014) Rapid approach to analyze biochemical variation in rat organs by ATR FTIR spectroscopy. Spectrochim Acta - Part A Mol Biomol Spectrosc 118:981–986. https://doi.org/10.1016/j.saa.2013.09.131
Sun H, Wang L, Zhang H et al (2019) Evaluation of yogurt quality during storage by fluorescence spectroscopy. Appl Sci 9(1):131. https://doi.org/10.3390/app9010131
Suzuki Y, Sato T, Sugimoto M et al (2017) Hydrogen-rich pure water prevents cigarette smoke-induced pulmonary emphysema in SMP30 knockout mice. Biochem Biophys Res Commun 492:74–81
Tang R, Ding C, Dang F et al (2018) NMR-based metabolic toxicity of low-level Hg exposure to earthworms. Environ Pollut 239:428–437. https://doi.org/10.1016/j.envpol.2018.04.027
Thannickal VJ, Fanburg BL (2000) Reactive oxygen species in cell signaling. Am J Physiol - Lung Cell Mol Physiol 279:1005–1028
Thomas MC, Woodward M, Li Q et al (2018) Relationship between plasma 8-OH-deoxyguanosine and cardiovascular disease and survival in type 2 diabetes mellitus: results from the ADVANCE trial. J Am Heart Assoc 7(13):17. https://doi.org/10.1161/JAHA.117.008226
Urbaniak SK, Boguszewska K, Szewczuk M et al (2020) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) as a potential biomarker for gestational diabetes mellitus (GDM) development. Molecules 25:202. https://doi.org/10.3390/molecules25010202
Vasantha-Srinivasan P, Senthil-Nathan S, Thanigaivel A et al (2016) Developmental response of Spodoptera litura Fab. to treatments of crude volatile oil from Piper betle L. and evaluation of toxicity to earthworm. Eudrilus Eugeniae Kinb Chemosphere 155:336–347. https://doi.org/10.1016/j.chemosphere.2016.03.139
Vollmer W, Blanot D, De Pedro MA (2008) Peptidoglycan structure and architecture. FEMS Microbiol Rev 32:149–167
Wang H, Chen J, Guo BY, Li J (2014) Enantioseletive bioaccumulation and metabolization of diniconazole in earthworms (Eiseniafetida) in an artificial soil. Ecotoxicol Environ Saf 99:98–104. https://doi.org/10.1016/j.ecoenv.2013.10.017
Wen S, Liu C, Wang Y et al (2021) Oxidative stress and DNA damage in earthworm (Eisenia fetida) induced by triflumezopyrim exposure. Chemosphere 264:128499. https://doi.org/10.1016/j.chemosphere.2020.128499
Whelan DR, Bambery KR, Puskar L et al (2013) Quantification of DNA in simple eukaryotic cells using Fourier transform infrared spectroscopy. J Biophotonics 6:775–784. https://doi.org/10.1002/jbio.201200112
William Lee Jolly (2020) Hydrogen. In: Encycl. Br. inc. https://www.britannica.com/science/hydrogen. Accessed 6 Jun 2020
Xin HG, Zhang BB, Wu ZQ et al (2014) Consumption of hydrogen-rich water alleviates renal injury in spontaneous hypertensive rats. Mol Cell Biochem 392:117–124. https://doi.org/10.1007/s11010-014-2024-4
Yesudhason BV, Kanniah P, Subramanian ER et al (2018) Exploiting the unique phenotypes of the earthworm Eudrilus eugeniae to evaluate the toxicity of chemical substances. Environ Monit Assess 190(3):145. https://doi.org/10.1007/s10661-018-6477-x
Yoneda T, Tomofuji T, Kunitomo M et al (2017) Preventive effects of drinking hydrogen-rich water on gingival oxidative stress and alveolar bone resorption in rats fed a high-fat diet. Nutrients 9(1):64. https://doi.org/10.3390/nu9010064
Yuan Y, Che L, Qi C, Meng Z (2019) Protective effects of polysaccharides on hepatic injury: a review. Int J Biol Macromol 141:822–830
Zhai X, Chen X, Lu J et al (2017) Hydrogen-rich saline improves non-alcoholic fatty liver disease by alleviating oxidative stress and activating hepatic PPARα and PPARγ. Mol Med Rep 15:1305–1312. https://doi.org/10.3892/mmr.2017.6120
Zhang J, Xue X, Han X et al (2017). Hydrogen-Rich Water Ameliorates Total Body Irradiation-Induced Hematopoietic Stem Cell Injury by Reducing Hydroxyl Radical. https://doi.org/10.1155/2017/8241678
Zhang QL, Dong ZX, Luo ZW et al (2020a) The impact of mercury on the genome-wide transcription profile of zebrafish intestine. J Hazard Mater 389:121842. https://doi.org/10.1016/j.jhazmat.2019.121842
Zhang X, Liu J, Jin K et al (2018) Subcutaneous injection of hydrogen gas is a novel effective treatment for type 2 diabetes. J Diabetes Investig 9:83–90. https://doi.org/10.1111/jdi.12674
Zhang Y, Liu Q, Yin H, Li S (2020b) Cadmium exposure induces pyroptosis of lymphocytes in carp pronephros and spleens by activating NLRP3. Ecotoxicol Environ Saf 202:110903. https://doi.org/10.1016/j.ecoenv.2020.110903
Zhu, J., Li, Z. G., Yang, D. L., & Mao JY (2011) Acute toxicological effects of Hg pollution on earthworm Eisenia foetida. ournal Shanghai Inst Technol (Natural Sci 2.
Author information
Authors and Affiliations
Contributions
All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by MK, MNA, AO, and MB. The first draft of the manuscript was written by MK, and all authors commented on previous versions of the manuscript. The supervision was performed by DA. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Communicated by Chris Lowe.
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Köktürk, M., Atalar, M.N., Odunkıran, A. et al. Evaluation of the hydrogen-rich water alleviation potential on mercury toxicity in earthworms using ATR-FTIR and LC–ESI–MS/MS spectroscopy. Environ Sci Pollut Res 29, 19642–19656 (2022). https://doi.org/10.1007/s11356-021-17230-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11356-021-17230-x