The study of the modulation of the toxicity of heavy metals by coexisting chemicals in the environment is vital for realistic ecological risk assessment. Our study was aimed at determining possible toxicity modulations of Cd by humic acid (HA) using the Allium cepa test system. A. cepa bulbs were exposed to Cd (1 and 5 mg/L) and HA (10 mg/L) individually or in mixtures. The root lengths of the bulbs and cytogenetic endpoints in root meristematic cells, including the mitotic index (MI), nuclear abnormalities (NAs), and chromosomal abnormalities (CAs), were determined. The results revealed that the MIs of A. cepa co-exposed to HA and Cd were significantly recovered by >15% compared with those of A. cepa subjected to Cd-only treatments, and this response was more sensitive than the phytotoxic response (root length). Furthermore, the burden of NAs was significantly decreased in the co-exposed bulbs by >20% compared with bulbs with Cd-only treatments. The frequencies of CAs were also reduced in the bulbs co-exposed to HA and 1 and 5 mg/L Cd by >15 and >25%, respectively, compared with bulbs receiving Cd-only treatments. Therefore, our findings indicated that HA plays a significant protective role in Cd toxicity in A. cepa.
Allium cepa was used to determine the modulation of Cd toxicity by humic acid using microscopic and macroscopic endpoints.
Humic acid induced the recovery of the inhibited mitotic index and root lengths caused by Cd stress.
The burdens of nuclear and chromosomal abnormalities were alleviated by humic acid.
Humic acid plays a protective role against Cd toxicity in the A. cepa test system.
The rare element cadmium (Cd) is a naturally occurring heavy metal in the Earth's crust. Natural phenomena such as weathering and volcanic emissions release Cd into the environment. However, the release of Cd into the environment has been accelerated by anthropogenic activities such as the use of phosphorus-based fertilizers, burning of fossil fuels, metal ore combustion, and its industrial application in batteries, paint pigments, metal alloys, and coatings (Bernhoft 2013; Jaishankar et al. 2014).
Cd is considered highly toxic to living organisms, including plants, animals, and humans. The ecotoxicity of Cd has been tested widely using several plant and animal species over the years (Das et al. 1997; Genchi et al. 2020). Furthermore, the International Agency for Research on Cancer (IARC) has classified Cd and Cd compounds as carcinogenic to humans (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans 2012). Cd causes cancer in animals by inducing mutations in critical genes and/or stimulating cellular signals, thus promoting cell proliferation. However, plant cells do not respond to Cd by stimulated cell proliferation (Deckert 2005). Despite this fact, upon exposure to Cd, both plant and animal cells show similar cytotoxic and genotoxic effects, and the latter may lead to mutagenicity (Seth et al. 2008; El-Habit & Moneim 2014).
Cytotoxic and genotoxic (cytogenotoxic) effects in an organism can be recognized as early events after exposure to harmful chemicals (Hagmar et al. 2001). Therefore, they are rapidly responding and sensitive endpoints in the assessment of the toxicity of contaminants/contaminant mixtures. Examples of some commonly evaluated cytogenetic endpoints in plant and/or animal test systems include the mitotic index (MI), chromosomal abnormalities (CAs), nuclear abnormalities (NAs), and the formation of micronuclei (MN) (Tucker & Preston 1996; Leme & Marin-Morales 2009). The MI is characterized by the total number of dividing cells in the cell cycle. Exceptionally high or low MIs indicate the cytotoxicity of an agent. CAs are characterized by changes in either chromosomal structure (due to clastogenic actions of harmful chemicals) or chromosome number (due to aneugenic actions of harmful chemicals). The CAs are widely used to detect the genotoxicity of agents. The NAs are characterized by morphological alternations in non-dividing nuclei, which denote the cytotoxic and/or genotoxic potential of an agent. On the other hand, MN, which represent the occurrence of additional small nuclear structures other than the main nucleus of a cell, are used to evaluate the mutagenicity of an agent (Tucker & Preston 1996; Hagmar et al. 2001; Leme & Marin-Morales 2009).
Previous cytogenetic studies showed that Cd can alter the MIs, induce CAs both by clastogenic and aneugenic actions and cause NAs and MN in different plant and animal test systems (Cavas et al. 2005; Çelik et al. 2005; Seth et al. 2008; Arya & Mukherjee 2014; El-Habit & Moneim 2014; Jaiswal et al. 2022). However, there is a vacuum in the empirical studies on the possible modulations in Cd-induced cytogenetic effects in organisms caused by co-exposure to other natural chemicals present in the environment, such as humic substances (HSs).
HSs are a group of ubiquitous, naturally occurring organic compounds in both terrestrial and aquatic environments. Approximately 60% of soil organic matter consists of HSs, and in freshwater environments, they make up about 50–80% of the dissolved organic carbon (DOC) at concentrations ranging from 1 to 100 mg/L (Steinberg et al. 2006; Shah et al. 2018). HSs originate from the degradation of plant and animal residues by microbiological, chemical, and photochemical processes (Rodrigues et al. 2009). Therefore, they have complex structures of heterogeneous organic molecules that have both hydrophilic and hydrophobic domains with different functional groups in the same structure (Piccolo 2001). This aids them in binding with both polar and nonpolar xenobiotics such that their bioavailability to organisms is altered. Therefore, a particular toxicological interest can be placed on HSs. Depending on the solubility under different pH conditions, HSs can be broadly classified into humic acid (HA, soluble under alkaline pH conditions), fulvic acid (FA, soluble under any pH condition), and humin (insoluble under any pH condition) (Ishiwatari 1992). Even though relatively little information is available on the genetic actions of HSs, there are few previous reports on their antitoxic and antigenotoxic, and/or antimutagenic properties over different xenobiotics evaluated using different biological test systems/bioassays (Ferrara et al. 2001; Loffredo et al. 2008; Marova et al. 2011; Shahid et al. 2012; Osina et al. 2022).
Among several plant and animal-based bioassays, the Allium cepa (common onion) bioassay is a highly effective and efficient plant bioassay for evaluating the cytotoxicity and genotoxicity of environmental contaminants (Leme & Marin-Morales 2009). The presence of clear mitotic phases, a reduced number of large chromosomes (2n = 16), and rapid responses make it an excellent model for detecting a number of cytogenetic endpoints including the MI, NAs, CAs, and MN. Moreover, findings from the A. cepa test system have a broader application as it is documented to show a good correlation with mammalian test systems (Fiskesjo 1985; Grant 1994; Leme & Marin-Morales 2009). Besides the microscopic observations of cytogenetic effects, the A. cepa bioassay can also be used to evaluate macroscopic phytotoxic endpoints such as root and/or shoot growth (Fiskesjo 1988). Therefore, the A. cepa bioassay has been frequently used as a sensitive test system in environmental monitoring for several decades.
In this context, we hypothesized that HA, as a substitute of HSs, can modulate the toxicity of Cd, and that this may be rapidly and effectively evaluated by assessing cytogenetic endpoints using the A. cepa bioassay. Even though the modulation of the toxicity of contaminants, including heavy metals, by HSs was previously assessed through different plant- and animal-based experiments, the use of cytogenetic endpoints to detect such modulations is relatively new and our knowledge on such early response modulations at molecular and genetic levels in biological systems is limited. Therefore, the aim of this study was to apply the A. cepa bioassay to determine the modulation of Cd-induced cytogenotoxicity by exposing A. cepa bulbs to Cd and HA alone and to their mixtures. Moreover, as an added toxicity endpoint, Cd-induced phytotoxicity and its modulation by HA were also evaluated by examining changes in root length. The findings of this study demonstrated a protective role of HA against Cd toxicity in A. cepa, unraveling a small part of the complex chemical reactions among multiple coexisting chemicals in the environment which change the consequences of exposure. Furthermore, the findings give insight into a potential remediation measure that can be applied in Cd-contaminated environments.
MATERIALS AND METHODS
Test chemicals and test solutions
Anhydrous cadmium chloride (CdCl2) (95% purity, first grade) and HA (practical grade) were purchased from FUJIFILM Wako Pure Corporation (Tokyo, Japan).
Two test concentrations of Cd (i.e., 1 and 5 mg/L) were selected on the basis of previous studies (Ünyayar et al. 2006; Seth et al. 2008; Arya & Mukherjee 2014; Jaiswal et al. 2022) that showed cytogenotoxic effects on the A. cepa test system to evaluate the modification of those effects (if any) by HA. A test concentration of 10 mg/L HA was selected to test the potential modifications of Cd-induced cytogenetic effects, on the basis of a preliminary experiment that showed statistically significantly higher root growth than that in the case of the control. The Cd and HA test solutions used in the experiment were prepared using Cd and HA stock solutions of 100 and 1,000 mg/L, respectively. Aerated tap water was used as the dilution water and as the control.
The stock solution of HA was prepared by dissolving 0.50 g of HA in 500 mL of 0.02 M NaOH (pH 12). The solution was kept on a magnetic stirrer for 2 days to facilitate thorough dissolution. The solution was then filtered through 0.7 μm glass fiber filters to remove suspended particles. The filtrate was used as the HA stock solution, which was stored in a glass bottle at 4 °C in the dark to prevent photochemical aging. At the time of the preparation of test solutions, the pH of all test solutions, both in the presence and absence of HA, including the control, was adjusted to 7.5 ± 0.1 using a few drops of 1 N HCl to prevent confounding effects of pH variability.
The actual Cd concentrations and the DOC contents in the test solutions prepared during the experiment were determined using an atomic absorption spectrometer (AAS) (Shimadzu AA 7000, Kyoto, Japan) and a total organic carbon (TOC) analyzer (TOC-2300, Hiranuma Sangyo, Ibaraki, Japan), respectively (Supplementary Table S1).
Equal-sized healthy A. cepa bulbs obtained from a local market were washed thoroughly with running tap water. After carefully scraping the bottom plates of the onion bulbs to expose the root primordia, they were placed over glass vials filled to the top with aerated tap water for 24 h in the dark at 25 °C. On the following day, onion bulbs with equal root lengths (∼1 cm) were exposed to the two concentrations of Cd (1 and 5 mg/L) alone or in combination with HA (10 mg/L). Another two sets of A. cepa bulbs were exposed to aerated water (control) and HA (10 mg/L). Each treatment or control had three replicates (n = 1 onion bulb per treatment), which were maintained separately in glass vials filled with 50 mL of the test solution or control water. The exposure setup was placed in an incubator (LH-55RDS, NK Systems Ltd, Tokyo, Japan) at 25 °C in the dark for 48 h with medium renewal after 24 h with freshly prepared test solutions. The water quality parameters (i.e., water temperature, pH, dissolved oxygen, and conductivity) in each test vessel were recorded at the beginning of the experiment and during medium renewal (Supplementary Table S2).
At the end of the exposure period, the length of the whole root bundle of each A. cepa bulb was measured as described by Fiskesjo (1993) by taking the measurement from the point where the roots sprout and down to where most root tips have ended their growth. The A. cepa bulbs were kept in the tube during this procedure to make it easier to obtain the vertical measurement by means of a ruler as recommended.
At the same time, several root tips (1–2 mm) from each A. cepa bulb were excised for the cytogenotoxicity assessment. The cytogenotoxicity assessment was carried out in accordance with the protocol developed by Grant (1982) with some modifications. Briefly, the root tips were fixed in Carnoy's solution (3:1 v/v, ethanol:glacial acetic acid) overnight. After fixation, the root tips were transferred to 70% ethanol and stored at 4 °C until microscopic observations. At the time of microscopic observations, the root tips were hydrolyzed in 1 N HCl at 60 °C for 7–8 min, washed with ultrapure water, and stained with acetocarmine for ≥15 min. The stained root tips placed on a glass slide with a drop of acetocarmine were squashed by applying gentle pressure to the coverslip to view a single cell layer. The cells were analyzed under a microscope (BZ-X810, Keyence, Osaka, Japan) for several cytogenetic endpoints to estimate the MI and the frequencies of NAs and CAs. The MIs (‰) were calculated as the number of cells undergoing different stages of mitosis (dividing cells) (i.e., prophase, metaphase, anaphase, and telophase) per 1,000 root meristematic cells in each A. cepa bulb. The frequencies of NAs (‰) were estimated by scoring different NAs in 1,000 interphase cells (non-dividing cells), and the frequencies of CAs (%) were estimated by scoring different CAs in a total of ∼100 cells undergoing metaphase, anaphase, and telophase in each A. cepa bulb.
The data, including the length of the root bundles, MIs, and the frequencies of NAs and CAs, were tested for normal distribution by the Shapiro–Wilk test. Since the data were found to be normally distributed, firstly, a one-way analysis of variance (ANOVA) and then Dunnett's post hoc test were carried out to identify the effect of each treatment (in the presence of Cd and/or HA) on each endpoint against the control group. Then the modulation of Cd-induced phytotoxic and cytogenotoxic effects by HA on each endpoint was determined by the independent samples T-test between the Cd-treated A. cepa bulbs and corresponding combinations with HA. A p-value <0.05 was considered statistically significant. All statistical analyses were performed with the IBM® SPSS® Statistics software (Version 25, IBM Corp., Armonk, NY, USA).
Apart from the main findings on the modulation of Cd-induced phytotoxicity and cytogenotoxicity in A. cepa root meristematic cells by HA, at the end of the 48-h exposure period, we observed the presence of suspended particles in the solution and particles that had been deposited on the roots and at the bottom of the vials in the treatment with 5 mg/L Cd and 10 mg/L HA (Figure 1), indicating a possible complexation reaction between Cd and HA that formed large particles.
Cd is a highly significant pollutant owing to its toxicity even at small concentrations and its high solubility in water. In the environment, its toxicity can be modulated by the presence of other chemicals such as HSs. HSs have been found to play a protective role against the toxicity of different contaminants, such as heavy metals (Shahid et al. 2012; Yigider et al. 2016; Yildirim et al. 2021; Osina et al. 2022), pesticides (Misra et al. 2000; Loffredo et al. 2008), plant growth regulator maleic hydrazide used in agriculture (Ferrara et al. 2001, 2004), and stress conditions such as high salinity (Bakry et al. 2014), using different plant-based test systems. However, most of these studies have employed the yield/biomass and/or accumulation of Cd in plant parts as endpoints in field experiments that could take several days to months to obtain results. Here, we studied the modulation of Cd toxicity by HA by assessing more sensitive cytogenetic endpoints in A. cepa root meristematic cells and the root length of A. cepa bulbs as a phytotoxic endpoint upon short-duration exposure.
Elongation of roots occurs when the apical meristematic cells near the root tip pass through the interphase and different mitotic phases to complete the cell cycle, proliferating the parent cells. Since the rate of cell proliferation is indicated by the MI, both the MI as well as the root length are indicators of root growth (Alaguprathana et al. 2022). Our results showed that Cd decreases the MI and also reduces the root length in a dose-dependent manner, indicating its cytotoxicity and phytotoxicity. This observation has been recorded in several studies carried out elsewhere with A. cepa and explained as a result of the interaction of Cd with the proteins essential for cell cycle progression (Seth et al. 2008; Wang et al. 2014). On the other hand, as we expected, HA enhanced the MI and stimulated root growth in A. cepa. There is empirical evidence that HSs can enter the cells and become engaged in plant metabolism by facilitating protein synthesis as well as DNA synthesis, which aids in the progression of the cells toward mitosis through the S and G2 phases of the cell cycle (Gorova et al. 2005). When A. cepa bulbs were co-exposed to Cd and HA for 48 h, the root length after the low-Cd–HA treatment was significantly recovered, while the root length after the high-Cd–HA treatment seemed not to be recovered. However, microscopic observations indicated that the MIs were significantly recovered in both cases, showcasing the protective role of HA against Cd-induced toxicity. From this standpoint, we believe that with an increased duration of exposure, there is a possibility for root lengths to recover even in the high-Cd–HA treatment. Moreover, these findings indicate that the microscopic MI is a more sensitive endpoint than the macroscopic measurement of root lengths under a short-term exposure.
The evaluation of NAs and CAs is a sensitive analysis of the actions of a test agent and hence, of any modulations (if present) upon co-exposure to multiple agents. In this study, both NAs and CAs were evaluated in the A. cepa test system after exposing bulbs to Cd and HA alone and in mixtures.
As observed in previous studies, the tested Cd concentrations caused significant occurrences of NAs including condensed nuclei, nuclear buds, binuclei, and MN, in the root meristematic cells of A. cepa bulbs. The most frequently observed NA in this study, the condensed nuclei with smaller or faded nucleoli and disintegrating cytoplasm, is an indication of the cell death process (cytotoxicity). Previously, Silveira et al. (2017) also observed condensed nuclei as the most prominent NA type upon Cd exposure in A. cepa root meristematic cells. Moreover, according to Leme & Marin-Morales (2009), polynucleated cells also indicate a cell death process. The MN demonstrate the mutagenicity of Cd, and they arise as a result of DNA damage, and not or wrongly repaired DNA in the parent cells affected by the impact of Cd. Other than this, they may also originate from an elimination process of excessive genetic material from the main nucleus (Leme & Marin-Morales 2009; Wang et al. 2014). Moreover, it is documented that the nuclear buds will also eventually develop into MN as a result of the aforementioned elimination process of excessive genetic material (Wang et al. 2014). The root meristematic cells of A. cepa bulbs exposed only to HA did not exhibit any significant NA other than the spontaneous NAs that appear similarly in the control bulbs. However, interestingly, the cumulative NA burden was significantly lowered by HA in the A. cepa bulbs co-exposed to Cd and HA compared with those of corresponding Cd-only treatments, portraying the anticytotoxic, antigenotoxic, and antimutagenic potentials of HA against Cd toxicity.
As in the previous findings of Cd-induced CAs in the A. cepa test system (Borboa et al. 1996; Seth et al. 2008; Arya & Mukherjee 2014; Jaiswal et al. 2022), our results demonstrated a clastogenic as well as aneugenic action of Cd on chromosomes as seen by the higher frequencies in the occurrence of CAs such as chromosome bridges and breaks, which are indicative of clastogenic effects, and chromosome losses, polar slips, and C-metaphases, which are indicative of aneugenic effects (Leme & Marin-Morales 2009). Among the observed CAs, the spindle-related abnormalities demonstrate that one of the primary actions of Cd is on mitotic spindles. However, the most frequently observed CA type in this study was chromosomal breaks. This may be associated with the ability of Cd to induce reactive oxygen species (ROS), which can damage DNA and proteins that are involved in processes including DNA replication and repair (Seth et al. 2008; Genchi et al. 2020). Moreover, Arya & Mukherjee (2014) reported that Cd induces oxidative stress in A. cepa root tissues, which is well correlated with CAs including DNA fragmentation and MN formation. Moreover, as explained above, one of the mechanisms by which MN may form is the inability to integrate the chromosome fragments into the daughter nuclei at the end of the completion of mitosis (Wang et al. 2014). On the other hand, HA alone did not exert any significant clastogenic or aneugenic activity on A. cepa chromosomes in this study. However, in the co-exposure treatments with Cd and HA, significant reductions in CA were observed compared with their corresponding Cd-only treatments, demonstrating the antigenotoxic potential of HA against Cd toxicity.
The observed protective effects of HA on the Cd-induced cytotoxicity, genotoxicity, and/or mutagenicity in the A. cepa test system can be explained by three possible mechanisms. The first is the reduction in the bioavailability of free Cd ions owing to their complexation with HA, preventing their absorption by the roots. Different functional groups, especially the carboxylic groups, are considered to be the major functional groups of HSs with which metal cations interact to form complexes (Spark et al. 1997; Liu et al. 2020). The Cd–HA complexes are probably too large to be absorbed by the roots. The second mechanism would be the participation of the HA molecules absorbed into the root tip cells in beneficial cellular processes such as the promotion of cell division by facilitating protein and DNA syntheses. The third mechanism would be the participation of HSs in stress tolerance pathways. For example, the fundamental cause of the observed NAs and CAs in the A. cepa root meristematic cells is the Cd-induced oxidative damage of cellular genetic material and/or proteins. Several authors suggest that HSs act as radical scavengers, especially when they contain electron-rich aromatic structures (Marova et al. 2011; Yigider et al. 2016). Moreover, HSs are associated with the biosynthesis of glutathione, which is one of the key antioxidants in organisms (Shah et al. 2018). Furthermore, a recent report by Cha et al. (2021) reveals that HA is involved in upregulating a diverse set of abiotic stress-related genes encoding heat shock proteins and redox proteins in Arabidopsis grown under salt stress. Moreover, genes encoding transcription factors involved in plant development and abiotic stress tolerance have also been upregulated by HA.
In this study, we demonstrated the protective role of HA against the toxicity of Cd by evaluating the modulation of sensitive cytogenetic as well as phytotoxic endpoints in the A. cepa test system. We believe that this protective role is attributed to the reduced bioavailability of free Cd ions due to their complexation with HA, thereby reducing the Cd uptake, and the HA-mediated molecular and biochemical pathways that are beneficial to plant growth and tolerance to abiotic stress, especially those related to oxidative stress. These findings also suggest that the application of HSs could be a potential remediation measure that can be applied in aquatic and terrestrial environments contaminated by Cd owing to industrial activities. However, the impacts of simultaneous exposure to Cd and HA in other organisms in such environments should be investigated before implementing this as a remediation measure.
We are grateful to the anonymous reviewers for their constructive suggestions in improving this manuscript.
T.S.: Conceptualization; Methodology; Investigation, Writing – original draft, T.F.: Conceptualization; Methodology; Investigation; Resources; Writing – review & editing; Supervision.
This study was partially supported by the Strategic Research Area for Sustainable Development in East Asia (SRASDEA), Saitama University.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
CONFLICT OF INTEREST
The authors declare there is no conflict.