INTRODUCTION:

Many substances that are involved in agricultural, industrial, cosmetic, and domestic products are released into the water environment¹. These substances are commonly toxic and life-threatening even at low concentrations greater than their natural levels and can cause harmful effects as a result of their bioaccumulation ^2-9. Cadmium is an environmentally hazardous metal that is found naturally at a low concentration, 0.1 - 0.5mg/kg dry weight in the earth's crust. Also, it has a long biological half-life ranged between (10 - 30) years makes it in the ranks of top of pollutants worldwide according to World Health Organization (WHO) and Agency for Toxic Substances and Disease Registry (ASTDR). Organisms of the aquatic environment usually absorb cadmium from their polluted habitat by digestive, respiratory, and body surface systems without secretion and tend to be affected by this hazardous heavy metal^10-12. Pollution of the environment with cadmium is a subject of concern that can result in the entrance of Cd into the food chain and lead to serious problems in the health of humans as well as other organisms^13,14.

Cd (II) ions can be adsorbed and removed from the aqueous environment using coffee powder or Indole functionalized polysulfone polymer under optimum conditions^15,16. The use of convenient lab animals as pollution indicators is a usual manner to understanding the plan of action of cadmium toxicity in many species of the biosphere. Numerous aquatic organisms are used as a model to investigate the effect of metal toxicity on metabolic processes, growth, and survival^17-19. Gastropod mollusks are an adequate indicator of contamination by metals, they promising bioindicators of heavy metals that is because they are highly tolerant to metals and the ability to accumulate large quantities of metals in their tissues^20-22. Mollusks have open blood circulating systems and this resulted in direct contact between their blood and the surrounding aquatic environment. Thus, mollusks are considered a pivotal index to detect water pollution as well as the health status of the organisms²³. There is a detrimental effect of toxic metal ions on the numerous blood parameters in the aquatic organisms, together with mollusks, as complexes resulted from these toxic metal ions with the structural proteins, nucleic acids, and enzymes that interfere with their normal vital functions^24,25.

MATERIALS AND METHODS:

The sample of this study, gastropod Lymnaea radix cor, was obtained from the Shatt Almadina stream that belongs to the Euphrates River in spring 2018. The site has North 30.95. 7688 and East 47.268223 longitude and latitude respectively. Samples were brought to the laboratory along with conserved freshwater from their location. The adult (10-12mm) animals, which were selected for the current study, were identified depending on ²⁶. In the laboratory, they were kept in a 10L container filled with stored tap water. And then, they were allowed to acclimatize to the conditions of the laboratory for 72 h before the beginning of the experiment under a 13 h light / 11 h dark performed at 25^oC using continuous aeration. Lettuce was used twice time in a day to feed snails and the stored tap water changed daily.

Experiment:

Exposure stock solution with a concentration (100mg/l) of cadmium chloride anhydrous CdCl₂, molecular weight 183.32g/mol -Thermo Fisher Scientific, was prepared using stored tap water. From this stock, series of concentrations (0, 20, 40, 60, 80, and 100µg/l) were prepared using stored tap water in 500ml exposure containers. The experiment started by adding 10 acclimatized animals in each exposure container. The same conditions of acclimatization, temp., light/dark, aeration and feed, were used during exposure periods (1, 3, and 5 days) and the exposure water was renewed daily.

Hemolymph Isolation:

At the end of exposure periods, hemolymph is needed to proceed with a comet assay for snails exposed to the cadmium chloride. In the first, the snail was wiped and the foot was then punctured by a micropipette to collect 100µl of the hemolymph that was kept into an Eppendorf tube of 1.5µl. and then stored in freezing until used in tests.

Comet Assay:

Oxiselect comet assay kit from Cell Biolabs, USA, was used to assess the DNA damage. The procedure was performed according to the manufacturer's instructions. As it's known, the principle of this method included a mix of cells sample (hemolymph in the current study) with a low melting point agarose and apply on an oxiselect comet slide covered with regular point agarose. The mixed cells are then applied for a lysis buffer to lysate the cells and eliminate proteins, cellular membranes, RNA, and cytoplasmic and nucleoplasmic constituents. Subsequently, the slides treated with an alkaline solution to denature and relax DNA. Lastly, the sample slides were electrophoresed to distinguish intact DNA from those damaged. The samples were stained with Vista green DNA dye and visualized under a microscope attached to an image analysis system. DNA damage was visually scored according to²⁷.

RESULTS AND DISCUSSION:

Although toxicity caused by metals was given wide attention, still unsatisfied knowledge about effects resulted from long-term exposure to low concentrations of metal. Comet assay, as a genotoxicity biomarker, is a useful method for early detecting commensurable changes in DNA integrity and other cellular parameters. Comet assay biomarker can give an impression about the existence of disturbance in the environment before it develops to transfer to an upscale of biological organizations^28,29. In the present study, Lymnaea radix cor, that collected from a site that belongs to the Euphrates river (Figure 1), was exposed to different concentrations of cadmium for different periods. The results showed that there is no DNA damage at all concentrations of cadmium exposure for one day. With an increase in exposure time, damage in DNA hemolymph existed. Significant differences in the DNA tail percentage showed at concentrations 60, 80, and 100µg/l of cadmium for 3 days of exposure time compared to the same concentrations resulted from one day exposure time (Figure 2). Furthermore, the DNA tail percentage increased at concentrations 60, 80, and 100µg/l of cadmium when exposure time raised to five days compared to the same concentrations resulted from three days exposure time (p≤ 0.05). And the highest percentage of DNA damage, (18%), recorded at 100µg/l of cadmium for the exposure time of five days. On the other hand, the results revealed that there are no significant differences in the DNA tail percentage at concentrations 20 and 40µg/l of cadmium regardless of exposure time.

Figure 1: Sampling location of Lymnaea radix cor from Shatt Al Madina stream of Basra.

Figure 2: Damage of DNA. Values are means ± Standard error of the tail DNA resulted from the comet assay applied on Lymnaea radix cor., indicate for exposure to a concentration 0, 20, 40, 60, 80 and 100µg/l of cadmium chloride respectively. Periods of exposure are 1, 3, and 5 days. The same letters indicate no significant differences (Duncan's Multiple Test Range).

Figure 3: DNA detected by comet assay in the hemolymph of Lymnaea radix cor. (a) Control cells; (b) Damaged cells resulted from exposure to cadmium chloride.

Many studies showed that the toxic effects of cadmium caused changes in behavioral, hematological, and biochemical parameters in freshwater snails^20,30,31. De Silva showed that the cadmium levels were highest in the viscera and foot muscle of estuarine mud snail Amphibola crenata exposed to toxic concentrations of waterborne cadmium, and this cd burdens resulted in declining of (O:N), energy imbalances, glycogen decreases and effect on oxidative stress³². The results of this study agreed with previous studies which revealed that the genetic material of snails affected by cadmium, Sarkar showed that the DNA tail (%) increased in the gill of marine gastropod snail Nerita chamaeleon exposed to different concentrations of cadmium chloride³³. Also, the current study is compatible with a study by Vincent-Hubert who’s showed that the Cadmium was genotoxic in embryos and neonates of the freshwater snail Potamopyrgus antipodarum exposed to cadmium, and the last could cause inhibit of DNA repair enzymes³⁴. A study by Nica, which also supports our study, showed that the genetic material of hepatopancreas of adult snail Cantareus asperses affected by exposure to cadmium which resulted in an increase in DNA methylation and influence on gene expression²¹.

CONCLUSION:

No references regarding genotoxicity of cadmium towards species Lymnaea radix cor from Iraqi aquatic environment. To conclude, the results of the current study showed a genotoxic effect in the hemolymph of the snail Lymnaea radix cor exposed to cadmium. Furthermore, the comet assay can be applied on Lymnaea radix cor and it easy, fast, and susceptible tool to detect the damage of DNA in the organism. In consequence, the freshwater snail Lymnaea radix cor is a useful candidate for genotoxicity and provides an early caution, biomarker organism, of contamination of the freshwater ecosystem by cadmium.

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Received on 25.04.2021 Modified on 26.10.2021

Research J. Pharm. and Tech. 2022; 15(6):2761-2764.

DOI: 10.52711/0974-360X.2022.00462