Herbicide pyrazosulfuron ethyl induces genotoxicity, cell cycle alterations and apoptosis in tilapia (Oreochromis mossambicus)

Abstract

Fishes are excellent subjects for the study of the mutagenic and carcinogenic potential of contaminants present in water. Herbicides are one of the toxic contaminants that cause lethal effects in fish. In this context, the present study was designed to study the sub‐lethal toxic effect (low dose [LD] 25 mg/L, medium dose‐50 mg/L, and high dose [HD] 100 mg/L) of a commonly used herbicide, pyrazosulfuron ethyl (PE), on erythrocytes of tilapia. The genotoxic effect was studied to understand the sub‐lethal potential by means of (a) the nuclear abnormalities, including the micronucleus (MN) frequency; (b) further, each stage of the cell cycle was analysed using flow cytometry and (c) finally, the targets of PE was deciphered using in silico analysis. The results showed that there were significant effects of time (7 and 14 days) and doses in the increase in the frequency of MN in erythrocytes of fish treated with PE, compared to the control group. After exposing fish to PE for 7 and 14 days, there was a proportional increase in the frequency of the apoptotic population of erythrocytes for LD to HD. During both time exposures (7 and 14 days), the highest cell death was accounted for in HD. Moreover, the majority of the cells showed a dysregulated cell cycle in the G1 phase in all the treated groups of PE, compared to the control group. This study showed that PE might initiate genotoxicity in non‐target species (tilapia) of the aquatic ecosystem near agricultural fields.

INTRODUCTION

The effective use of herbicide is a major challenge in forestry and sustainable agricultural management. Pesticides might reach the aquatic ecosystem by runoff, soil leaching, aerial drift or inadvertent overspraying during dispersion treatments, causing harm to freshwater ecosystems ( Lushchak et al., 2018). There is critical concern about the genotoxicity of pesticides used in agriculture. As an important part of the natural environment, the freshwater ecosystem faces the threat of a diminishing genetic base and biodiversity because of the indiscriminate use of pesticides (Guilherme et al., 2014; Omitoyin et al., 2006; Relyea, 2005).

Several species are used as models for studying the genotoxic potential of environmental toxins, as they can metabolize, concentrate and store xenobiotics in their system (Shoaib & Siddiqui, 2015; Shoaib et al., 2013). Thus, fish like Mullus sp., Platichthys sp. and Oreochromis sp. are used as test models in surveillance systems (Naqvi et al., 2016). Moreover, genotoxic chemicals are responsible for DNA damage, causing malignancies and reducing the survival of adult fishes (Scalon et al., 2010).

Pyrazosulfuron ethyl (PE) is rampantly used in agricultural fields, resulting in residues in surface water and groundwater through runoff and leaching, but is less studied to the best of our knowledge concerning the mechanisms behind its potential DNA-damaging action. In addition, the half-life of PE has been observed in the near-aquatic environment by agricultural runoff (Singh & Singh, 2011). In line with this, previous studies performed by our group (Pandya & Parikh, 2016; Pandya et al., 2018; Upadhyay et al., 2014) have provided deeper insights into neuroendocrine toxicity at the expression level (gene and protein) and general biochemical alterations. Oreochromis mossambicus was selected as the test model due to its abundance in the freshwaters of India and its sturdiness (Moyle et al., 2010). Therefore, the genotoxic potential of PE was investigated through the assessment of the development of nuclear abnormalities, micronucleus (MN), cell cycle arrest, and cell death.

MATERIALS AND METHODS

Experimental design

Male and female adult O. mossambicus were obtained from pure brooders with a length of 12 ± 3 cm and weight of 25 ± 3 g. Fish (five males and five females) were acclimated in a clean glass tank for 15 days in de-chlorinated water at 27 ± 4°C, pH 7.4 ± 0.05, dissolved oxygen 8 ± 0.3 mg/L and total hardness 188 mg/L CaCO₃ with a 12:12 light: dark photoperiod according to Pandya et al. (2018). If in any batch mortality exceeded 5% during acclimatisation, that entire batch was discarded. After the acclimation period, a total of 40 fish were divided into four groups with five females and five males (n = 10) in each group, and three replicates were performed. Sub-acute doses were calculated based on the LC₅₀ value (500 mg/L) after 96 h (Upadhyay et al., 2014); sub-acute dose 1/20th LC₅₀ (low dose [LD]), 1/10th LC₅₀ (medium dose [MD]) and 1/5th LC₅₀ (high dose [HD]) were chosen for health assessment protocol studies. They were divided into four groups: (1) Control, (2) LD-25 mg/L, 3. MD- 50 mg/L, 4. HD-100 mg/L of herbicide PE (Saathi-10% wettable powder (WP)) obtained from previously established LC₅₀ value for 7 and 14 days. The blood was collected using the tail ablation method using a heparinised syringe and was kept at −20°C for further experimentation (Upadhyay et al., 2014). The PE was dissolved in water, and a semi-static system was maintained, in which half of the water (20 L) was replaced every three days and an equal amount of the PE was added (Upadhyay et al., 2014). All the groups were kept under continuous observation during the experimental period. Commercially available food pallets were given to fish once a day during the experiment ad libitum.

Nuclear abnormalities and MN detection

Blood was drawn from the control and experimental groups and placed on clean glass slides to make a thin smear. The slides were kept on ice-cold condition for 2 h, fixed in methanol for 10 min, and stained in 10% Giemsa (Sigma G5637). MNs were scored in one thousand cells from each slide and were identified as small non-refractive, circular or ovoid chromatin bodies separated from the central nucleus having similar stain like the main nucleus. The whole process was done in three replicates for each group. While the other abnormalities were checked according to the phenotypic characteristics, that is, presence of a notch, blebbing, formation of a lobe and dual nucleus (Piancini et al., 2015).

Cell cycle analysis

Cells (treated and untreated) were washed twice with cold phosphate-buffered saline and then suspended in 100 μl of 1X binding buffer (1 × 10⁶ cells in a 5-ml fluorescence-activated single-cell sorting (FACS) tube). Furthermore, 5 μl of fluorescein isothiocyanate (FITC) Annexin V (A13199, Thermo Fisher) and 5 μl eBioscience propidium iodide (PI; BMS500PI, Thermo Fisher) were added to 100 μl cells. The cells were vortexed and incubated for 15 min at RT (25°C) in the dark. Then, 200 μl of 1X binding buffer 1 M (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl₂) was added to each tube and analysed by flow cytometry (BD FACS Aria III) within 1 h. Unstained cells, cells stained with Annexin V (no PI compensation control), and cells stained with PI (no FITC Annexin V compensation control) were used as a control, and the parameter was repeated three times for each group.

In silico pathway analysis

Apoptotic and cell cycle regulatory gene network maps were generated using bioinformatics pathways via Pathway Common and WikiPathways and were visualised in the open-source software platform Cytoscape 3. The genes pertaining to the cell cycle and corresponding to zebrafish were taken into consideration, and the remaining undiscovered genes were eliminated.

Data analysis

Data were subjected to two-way analysis of variance to determine the effect of treatment as well as the period of treatment. A post hoc Dunnett multiple comparison test was conducted to test the significant differences between control and treated animals using GraphPad Prism 7 software.

RESULTS

MN quantification

The frequency of MN observed was found to be progressively increased in a range of 0.2 ± 0.06 to 0.8 ± 0.04 for 7 days and 0.9 ± 0.04 to 1.4 ± 0.01 for 14 days, compared to the control (0.1 ± 0.02 to 2.7 ± 0.62) in a dose- and time-dependent manner as summarised in Supplementary Tables 1 and 2. A direct proportion of erythrocytes with a higher MN frequency was seen in all the treated groups, compared to the control, which was found to be statistically significant (p < 0.05) for 7 and 14 days of sub-lethal MD and HD exposure. Furthermore, the number of nuclear abnormalities like lobed, notched, blabbed and bi-nucleated nucleus was higher with HD at 7 and 14 days, compared to the control (Figures 1–3) and was statistically (p < 0.05) significant at HD of 14 days, compared to 7 days’ treatment. Thus, it can be noted that the study had a higher incidence of other nuclear anomalies, compared to micronuclei in the erythrocytes examined.

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Cell cycle analysis

FACS analysis showed dose- and time-dependent changes in the cell proliferation and cell death of the erythrocytes. The cells were primarily in the G₁ phase of the cell cycle on Days 7 and 14 in all the exposed group fishes. The 7-day exposure showed a significant (p < 0.05) decrease in LD-, MD- and HD-exposed fishes, compared to the control (Figure 4). Conversely, 14 days of exposure resulted in a nonsignificant increase in MD-exposed fishes, compared to LD and HD.

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Moreover, the analysis of the synthesis phase (S phase) illustrated a significant (p < 0.05) increase in cells in the S phase of the cell cycle in LD-, MD- and HD-exposed erythrocytes, compared to untreated cells at 7 days (Figure 4). Among the treated groups, the 7’-day treatment determined that LD and MD displayed a significant increase (p < 0.05) in S phase cells, compared to the control and HD, which was not evident on the 14-day treatment. In G₂-M phase, erythrocytes were found to be significantly (p < 0.05) lower in the dose- and time-dependent manner. However, MD-exposed cells at 7 days were found to be increased (non-significant) in this phase, compared to control, LD and HD-exposed cells. A similar trend was observed on the 14 days of exposure, where there was a significant decrease in all the treated groups compared to the control (Figures 4 and 5).

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Apart from cell cycle analysis, apoptosis was also analysed among the treated groups. Apoptosis was found to be significantly (p < 0.05) increased after 7 and 14 days of exposure in all the treatment groups, compared to the control (Figure 4, Supplementary Figures 1 and 2). The highest percentage (40%) was found in HD-exposed cells of both durations, compared to the control.

In silico bioinformatics analysis

The next approach was to study the candidate markers' interrelation playing a role in the cell cycle and apoptosis. Therefore, the in silico bioinformatic approach was used for map generation using pathway commons. Apoptotic genes (bax, bcl2, apaf1, casp3 and casp 9) and cell cycle genes (cyclin A1 and cdk 1) were taken into consideration for the pathway construction, and the predicted targets for PE are represented in Table 1. The pathway common network analysis resulted in a total cluster of 559 genes (Figure 5), which were found to have a close grid and play a crucial role in the cell (Supplementary Table 3). Moreover, the results also revealed that apoptosis induction and alteration of the cell cycle indicated that PE has multiple target actions in this pathway. In total, four, five, six, nine and 10 genes were majorly found regulated by bax, bcl2, apaf1, casp 9 and casp 3, respectively. The remaining majority of genes were closely associated with cyclin A1 (ccna 1) and cdk 1. Deciphering the role of all genes in the pathway, 36 genes were assigned to have function for controlling state change, three were involved in controlling the expression, two were modifying the phosphorylation state, and two were involved in transport. In comparison, 445 other genes were found to be in close association and are assigned for complex formation.

TABLE 1 The possible pyrazosulfuron ethyl (PE) targets that were obtained from multiple screening from pathway common

Herbicide	Effects	Gene name	Known chemical	Chemical Entities of Biological Interest (CHEBI) ID:
PE	Alteration (Increase/Decrease)	bax	Azide	22680
			Imidacloprid, insecticide	5870
			Glyphosate, herbicide	27744
		casp3	Acetylsalicylic acid	15365
			Resveratrol	27881
			Cisplatin	27899
			Glyphosate, herbicide	27744
		casp9	Cisplatin	27899
			Glyphosate, herbicide	27744
		apaf1	Cisplatin	27899
			Glyphosate, herbicide	27744
		cycs	Cisplatin	27899
			Glyphosate, herbicide	27744
		ptk2	Cisplatin	27899
			Glyphosate, herbicide	27744
		sod2	Cisplatin	27899
		bcl2	Glyphosate, herbicide	27744
		ccna1 (cyclin A1)	Atrazine	15930

DISCUSSION

Studies on insecticides are available where they have proven the formation of MN in the erythrocyte of teleost (Katsumiti et al., 2009; Piancini et al., 2015; Ventura et al., 2008; Vryzas et al., 2011). However, studies are at their basal level when the effect(s) of herbicides are taken into consideration. Hence, the herbicide PE was selected for the present study to assess the genotoxicity. The basis for the selection was its routine and rampant usage, with fewer studies carried out on its toxic potential. As observed in the results, the DNA damage was triggered off in erythrocytes of O. mossambicus due to PE exposure at different concentrations, suggesting that PE possesses genotoxic potential and mutagenic properties. The control fishes had their intact DNA; thus, damage can be said to be a result of the clastogenic action of the PE.

Environmental mutagen has been reported to increase both micronuclei and DNA mutagenicity in fish (Russo et al., 2004). The first mechanism of action of genotoxic agents has been reported to be the promotion of DNA damage, resulting in three outcomes: the damage can be reversed, the damage can become permanent or the failure can lead to cell death (Vicari et al., 2012). Since MN and the central nucleus are usually released, their presence would mean their origin in a more recent cell cycle (Chandra & Khuda-Bukhsh, 2004; Graziela et al., 2010; Srivastava & Singh, 2015). Therefore, the most significant micronuclei frequency was also observed on Day 14, showing the greatest influence of PE on chromosome breakage, seemingly suggesting that this can induce genotoxic damage in erythrocytes of O. mossambicus. The research postulates that a long exposure period can lead to a decrease in MN formation intensity due to the formation of other related nuclear abnormalities associated with severe chromosomal aberrations (Bhatnagar et al., 2016; Karaismailoglu, 2015; Yadav & Jaggi, 2015). To overcome the toxicant stress and stabilise the micronuclei frequency, fish tend to reduce the frequency of the micronuclei and thereby promote a defensive mechanism (Bhatnagar et al., 2016; Braham et al., 2017; Da et al., 2011). Various studies on erythrocyte nuclear abnormalities have been proven to signal cytogenetic damage in fish species (Kousar & Javed, 2015; Strunjak-Perovic et al., 2009). In the present study, we also reported a significantly increasing trend of different types of nuclear abnormalities during the exposure time.

Among them, the maximum frequency was reported for the lobed nucleus. PE belongs to the sulphonylurea group and may induce mutation by virtue of its alkylating activity (Anbumani & Mohankumar, 2011). Thus, this unique property of alkylation may probably lead to cause DNA damage resulting in the formation of micronuclei and other nuclear abnormalities observed (Ayanda et al., 2018; Nwani et al., 2013; Vasanthi & Pechiammal, 2017). Our results are consistent with the results obtained by Ateeq et al. (2002), Barsiene et al. (2006) and Könen and Cavaş (2008) who discovered a significant increase in the MN frequency scale in erythrocytes of different teleosts.

The cell cycle analysis and apoptosis rate were performed with the help of FACS. This study stands at par with other conventional studies, as it analyses the direct effect of PE on the cells. The decrease in the G₁ phase is also attributed to a reduction in the cell cycle's overall doubling time. PE may act on cyclin-dependent kinases (cdk 4/6) and their cyclins (cyclin D), which results in a quick transition from G₁ to S phase. The possible trend obtained was in both the duration and was inversely proportional to the dose exposed.

For the S phase, there was a significant rise in LD and MD exposure at 7 days of PE exposure. The prominent steepness may be due to the burden caused by the pesticides during the synthesis of DNA and the time taken to repair, maintain and conserve the sequence. Moreover, it may have induced the expression of cyclin A and cdk 2, or in the gap, it may have decreased the expression of cyclin E. (Corlu & Loyer, 2012; Liu et al., 2007; Yabu et al., 2001). However, this was not observed in the case of 14 days of exposure, suggesting that adaptation or, more precisely, sequence alteration (mutation) might be induced, consequently normalising the S phase duration that was found to be similar to that of the control. The mutation(s) that may be induced by the exposure of PE can be point/insertions/deletions (InDels), signifying the activity of alkylating agents (Kondo et al., 2010; Rocco et al., 2010, 2011; Zhiyi & Haowen, 2004).

Further, the study proves that there is a reduction in G₂ to M phase in the 7- and 14 days’ exposed groups. This reduction attributes to the operation of the following mechanisms. First, PE may affect the activity of cyclins (cyclin A and cyclin B)- and cdk 1, which are the guardians (checkpoints) from the G₂ to M phase. Second, the mutations caused in the S phase may lead to misfolding of stress proteins and deactivation of chaperones, thus resulting in a reduction in the duration of the phases. These probable postulations explain the alarming condition of tilapia erythrocytes under PE exposure.

Thus, the MN studies and FACS results postulated that erythrocytes have entered apoptosis; together, these data show that PE exposure is potentially hazardous to O. mossambicus, even at sub-lethal levels as evidenced by the cell death study, which was found to be significantly increased in a dose- and time-dependent manner. At 7 days’ exposure, there was linearity in the increase in erythrocytes’ apoptotic population, that is, from LD to HD. This linearity was also observed for the 14-day exposure period. The highest cell death was accounted for by HD for both durations, suggesting that cells were under stress, which led them to undergo apoptosis at sub-lethal PE exposure.

As in the pesticide database until now, there is no record concerning the plausible targets of PE due to its herbicidal action. We evaluated the cell cycle and apoptotic pathway using a bioinformatics platform like pathway common, WikiPathways. Moreover, the MN and FACS data suggested that there is an alteration in the cell cycle genes. Therefore, the in silico cell cycle and apoptotic genes were extrapolated. The predicted pathway revealed a total of 559 genes that were found to be in a close association with the candidate genes (bax, bcl2, casp 3, casp 9, apaf 1, cdk 1, cyclins), which are either the transcriptional factors or the kinases that are involved in cell cycle regulation. Hence, we also predicted the potential targets of different compounds extracted from European Molecular Biology Laboratory (EMBL) and compared them with PE, suggesting that these are key targets modified by the herbicide (Table 1, Supplementary Table 3). As the maximum change observed was in MD and HD, we also predicted that the variations occurring in the gene expression pattern of these proteins would also be significant at both doses. This is because of our previously published data (Pandya et al., 2020), where we have shown a strong link between agrochemicals and endocrine markers, that is, hormones and their receptors are possible targets for agrochemicals, resulting in their multitarget competence. Nevertheless, the molecular structure analysis of PE through in silico applications and Gas Chromatography and Mass Spectrometry (GC–MS) studies will add to the present knowledge.

CONCLUSION

The present study established a base of the genotoxic potential of PE in O. mossambicus among the group of sulphonylurea. It also further proves the suitability of nuclear abnormalities, MN and FACS as tools for evaluating toxicity. The increase in the intensity of MN and other nuclear abnormalities with regard to cell death suggests the severity of PE on erythrocytes of tilapia exposed in a dose- and time-dependent manner. Moreover, cells were found to be undergoing apoptosis on 14 days of PE toxicity. The toxicity of the herbicide to O. mossambicus provides a basis to project the potential harm that may be caused to other inhabitants of the aquatic ecosystem and those who depend on them. Therefore, it may be imperative to ensure careful, efficient use of PE to prevent adverse effects, as this directly acts on the genome of the fishes and poses a risk to humans.

AUTHOR CONTRIBUTION

All the authors have worked immensely to give a desired shape to the article. Ankur Upadhyay helped with data curation, investigation, methodology validation, writing–original draft; Parth Pandya has done conceptualisation, methodology, writing–original draft, writing–review and editing; Ankita Salunke has helped in formal analysis and investigation, and Pragna Parikh has done resources supervision, writing–original draft, writing–review and editing.

ACKNOWLEDGEMENT

The authors are highly thankful to the Head of the Department of Zoology, the M.S. University of Baroda, Vadodara for giving facilities to carry out effective work.

CONFLICTS OF INTEREST

The authors declare there is no conflict of interest.

DATA AVAILABILITY STATEMENT

Data are available upon request from the authors.

ETHICS STATEMENT

As the edible fishes were used from the pure brooders, no ethical permission was required. All the experimental protocols were in accordance with the standard ethical procedures and guidelines of A.P.H.A., A.W.W.A. and W.P.C.F.

PEER REVIEW

The peer review history for this article is available at

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