Biochemical And Histopathological Responses Of Biomphalaria Alexandrina To RIPEX (plant Growth Regulator)Ⅰ

Abstract 

Background 

Plant growth regulators are widely used in agriculture for increasing the growth and ripening of plants, but they endanger the aquatic ecosystem. The current study assessed the effect of sublethal exposure to RIPEX 48% EC concentrations (8 and 16 µL/L) on oxidative stress parameters, sex hormones, immune potential enzymes, different‑ tial hemocyte counts, and the histopathology of digestive glands and ovotestis in Biomphalaria alexandrina snails. 

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Results 

RIPEX exposure caused an overall increase in Superoxide dismutase and Glutathione-S-Transferase activities in B. Alexandrina. However, extreme RIPEX exposure inhibits SOD activity in snails. Malondialdehyde activity showed an increase in B. Alexandrina exposed to both concentrations after all exposure periods. RIPEX also caused a significant increase in testosterone in snails exposed to 16 µL/L, it did, however, reduce hormone levels in snails exposed to 8 µL/L at 7 days. Regarding estradiol, there was a significant increase after 3 days of exposure to 16 µl/L and 7 days of exposure to 8 µL/L. RIPEX exposure also increased the activities of Myeloperoxidase and Adenosine deaminase enzymes in the digestive glands of snails. It increased the total hemocyte count of exposed snails as well as the number of granulocytes. Snail digestive glands and ovotestis showed pathological alterations after 7 days of RIPEX exposure. 

Conclusions 

These findings suggest that RIPEX is toxic to B. Alexandrina and that this snail can be used as a bioindicator for environmental contamination with plant growth regulators. 

Keywords 

Biomphalaria alexandrina, RIPEX, Testosterone, Estradiol, Myeloperoxidase, Adenosine deaminase, Histopathology

1 Background 

Agrochemical pollution of surface waters is a growing global environmental concern, particularly in areas where agriculture is rapidly expanding and intensifying. Various synthetic chemicals, such as pesticides or fertilizers, are used to modify crop quantity and quality to satisfy customers’ needs [7, 40]. Plant growth promoters are common agrochemicals in many countries [18]. 

When these chemicals are directly inhaled or consumed with contaminated vegetables, they hurt human health [39]. The organophosphorus pesticide, ethephon (2-chloroethyl phosphonic acid) is an ethylene-releasing synthetic chemical that is commonly used as a plant growth regulator (PGR) to promote fower induction, fruit ripening, and other physiological reactions. It is approved for use on a variety of food, feed, non-food, greenhouse nursery stock, and ornamental plants grown outdoors [3, 50]. Laboratory assessment of ethephon toxicity demonstrated that ethephon induced morphometric malformations of the second antenna, sensory bristles, tail spine, and rostrum of Daphnia magna which led to loss of function and death [54]. 

Abd-El Azeem [1] recorded a reduction of growth rate, DNA damage, and apoptotic efects in digestive gland cells of the slug Deroceras reticulatum after exposure to ethephon. Many authors approved the toxicity of Ethephon on rats and birds [2, 17, 55]. Furthermore, ethephon exposure caused genotoxicity, oxidative stress, and reproductive toxicity in mice [16]. Teratogenicity and gonadal system dysfunction in mice have also been linked to ethephon exposure [2, 16]. Ethephon has also been shown to be immunotoxic, have mutagenic efects [26], increase oxidative stress, and lower deoxyribonucleic and Ribonucleic acids concentrations [4]. 

Ethephon in water bodies may endanger aquatic fauna; however, data on its impact on aquatic organisms are scarce. Several aquatic species have been used to detect plant growth hormones in aquatic systems. However, some of these organisms are challenging to manipulate and maintain in the lab, making them more difficult to use in ecotoxicological evaluations [54]. Aquatic mollusks, including snails, have recently gained attention as test organisms due to the need for developing invertebrate models for the environmental monitoring of aquatic contaminants [21, 45]. Snails are the most abundant mollusk group in aquatic systems. Snails present a less exploitative alternative to higher vertebrates as bioindicators because they can be used in a variety of experiments without seeking ethical approval, and their acquisition and maintenance are inexpensive [13]. 

In this respect, Biomphalaria alexandrina snail has been widely used as an indicator organism of freshwater pollution with pesticides [29, 30]. Beyond its potential as a bioindicator, this snail acts as an intermediate host for the transmission of Schistosoma mansoni [35]. Therefore, agrochemical concentrations in the environment can influence schistosomiasis transmission through direct and indirect efects on the intermediate host and parasite densities [27]. The present investigation aimed at investigating the impact of sublethal exposure to the plant growth regulator, RIPEX 48% EC (the trade name of ethephon, 2-chloroethyl phosphonic acid) on various biochemical, immunological, and histological aspects of B. alexandrina snails as a bioindicator organism.

2 Methods 

2.1 Test organism 

Biomphalaria alexandrina snails were collected from Lake Manzalah located in El-Matareya, Dakahlia Governorate, Egypt (31° 11′ N 32° 2′ E) and maintained in well-aerated tap water at a constant temperature of 20 °C. They were fed fresh lettuce leaves daily. After maintenance for 4 weeks under the laboratory conditions, healthy mature snails (10–12  mm in shell diameter) were used in the subsequent experiments.

2.2 Experimental material and exposure conditions 

The plant growth regulator, RIPEX (a solution form containing 48% ethephon [2- chloroethyl phosphonic acid (C2H6ClO3P)] and 52% inert ingredients) was purchased from Chema Industries (New Nubaria city, Behira, Egypt; registry license No. 1840). A series of concentrations were prepared. Tree replicas with ten snails each were exposed. After 1-day of exposure, dead snails were counted, and removed and LC50 and LC90 were computed by statistical software of social sciences (SPSS; IBM Corp. Armonk, NY, USA).

2.3 Experimental design 

Two sublethal concentrations representing LC10 and LC20 were used (8 and 16  µL/L). 90 snails were divided into three groups, each group with three replicates (ten snails/replicate): the control group, the 8  µL/L-exposed group, and the 16  µL/L-exposed group. At 1-, 3-, and 7-day intervals, oxidative stress biomarkers and immune potential enzymes were assessed in the digestive gland. steroid hormones were assessed in Ovotestis. Differential hemocyte count, and histology of the digestive glands and ovotestis were assessed. For biochemical investigations, 0.5 g of tissue (digestive glands or ovotestis) from each group was homogenized in 0.5 mL phosphate buffer (pH 6.5) using a glass homogenizer and centrifuged at 10,000×g, 4  °C for 10  min. The resulting supernatants were used for subsequent assays.

2.4 Determination of oxidative stress markers 

2.4.1 Superoxide dismutase (SOD, EC 1.15.1.1) 

SOD activity was determined using the EnzyChrom™ Superoxide Dismutase Assay Kit (Cat. No: ESOD-100; BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions. The tissue homogenate using cold lysis buffer (50 mM potassium phosphate, 0.1  mM EDTA, and 0.5% Triton X-100) was centrifuged at 12,000g for 5  min at 4  °C, and the supernatant was collected for determination of SOD [43]. The activity of SOD was measured at the optical density (OD) at 440 nm.

2.4.2 Glutathione‑S‑Transferase (GST, EC 2.5.1.18)

Tis enzyme catalyzes the GSH conjugation with 1-chloro-2,4-dinitrobenzene (CDNB). It is measured spectrophotometrically at 340  nm. The reaction mixture contains Potassium phosphate buffer 0.1  M, pH 6.5, GSH dissolved in the potassium phosphate buffer (140  mg/100  mL), and CDNB 4  mg/ml in ethyl alcohol. One unit of enzyme activity is defined as the amount of enzyme which catalyzes the formation of 1 μmole of S-conjugate per minute [11].

2.4.3 Malondialdehyde (MDA) 

Malondialdehyde in whole homogenates was determined as thiobarbituric acid reactive substances (TBARS) according to the standard method of Ohkawa et al. [37]. The reaction mixture included 0.1 mL of the tissue homogenate, an equal volume of Sodium Dodecyl Sulfate solution, 0.75 mL of acetic acid, 0.75 mL of thiobarbituric acid, and 0.3 mL of distilled water. These components were mixed in a vortex and incubated in a boiling water bath for 1 h then cooled to room temperature. Ten 0.5 mL of distilled water and 2.5 mL n-butanol were added to each tube and vigorously mixed with a vortex then rotated in a centrifuge at 2,500×g for 10 min. Absorbance was read at 532 nm.

2.5 Hormones determination 

2.5.1 Testosterone 

Testosterone was determined using a testosterone Enzyme-linked immunosorbent assay (ELISA) kit (Cat. No: ADI-900-065; Enzo Life Sciences, NY, USA), Testosterone is the main androgen that efects both primary and secondary sexual development as sex drive. 100 µL of standard diluent (assay buffer 3 or tissue culture media) was pipetted into the non-specific binding and the maximum binding (0  pg/mL standard) wells. After that 100  µL of standard #1 through #5 into the appropriate wells. The sample (100 µL) was pipetted into the appropriate wells and 50 µL of assay buffer 3 into the non-specific binding wells. Also, 50  µL of yellow antibody were added into each well, except the blank, total activity, and non-specific binding wells. The incubation of the plate at room temperature on a plate shaker for 1 h at 500 rpm. After that, 50 µL of the blue conjugate was added into each well, except for total activity and blank wells. After emptying the wells from the contents, washing was done by adding 400  µL of wash solution to every well 3 times. Blue conjugate (5 µL) was added to the total activity wells and 200 µL of a solution of p-nitrophenyl phosphate was added to every well and incubated at 37 °C for 1 h without shaking. Reading of the plate at 405 nm was carried out after adding 50 µL of stop solution to every well [12, 48].

2.5.2 Estradiol 

Estradiol was determined using an estradiol ELIZA kit (Cat. No: 501890; Cayman Chemical, Michigan, USA) according to the manufacturer’s protocols. The principle of this reaction is based on that concentration of estradiol acetylcholinesterase (AChE) tracer is constant, but the native estradiol concentration varies therefore the amount of estradiol acetylcholinesterase (AChE) tracer which binds to estradiol antiserum is inversely proportional to the concentration of native estradiol in the well. 100 µL ELISA buffer was added to non-specific binding and maximum binding wells. Addition of 50 µL from tube #8 to both lowest standard wells (Standard 8) and 50 µL from tube #7 to each of the next standard wells (Standard 7). A sample (50 µL) was added to each well. Estradiol AChE Tracer (50 µL) was added to each total activity and blank wells. After that Estradiol ELISA antiserum (50 µL) was added to each well except for total activity, non-specific binding, and blank wells. After orbital shaker incubation (2 h at room temperature), absorbance was measured at 414 nm [52].

2.6 Determination of immune potential enzymes 

2.6.1 Myeloperoxidase (MPO, EC 1.11.2.2) 

One unit of MPO activity is defined as the amount of enzyme that hydrolyzes the substrate and generates taurine chloramine to consume 1.0 μmole of TNB per minute at 25 °C. MPO was determined using a myeloperoxidase colorimetric activity assay kit (Cat. No: MAK068; Sigma-Aldrich, MO, USA). The absorbance was measured at 412 nm.

2.6.2 Adenosine deaminase (ADA, EC 3.5.4.4) 

ADA is an enzyme that catalyzes the conversion of adenosine and 2’-deoxyadenosine to inosine and 2’-deoxyinosine. ADA was determined according to the principles of the technical bulletin adenosine deaminase activity assay kit (Cat. No: MAK400; Merck KGaA, Darmstadt, Germany). One unit of adenosine deaminase is the amount of enzyme that hydrolyzes adenosine to yield 1.0 μmol of inosine per minute at 37  °C. The reagents are 1×ADA assay buffer (41 μL), ADA Converter (2 μL), ADA developer (2 μL), and ADA substrate (5 μL).

2.7 Differential hemocyte count 

The hemolymph was collected from the snail (control, exposed) according to Sminia [47]. After being touched on the head-foot by a Pasteur pipette, the snail was forced to retract deeply into its shell and extrude hemolymph, which was collected for a differential hemocyte count. Hemolymph was smeared on the microscope slide for spreading. After fixation in 99.8% methanol for 5 min, the slide was turned at a 45° angle for drying at room temperature and stained with Giemsa stain for 20  min. The differentiation of hemocytes was determined to be proportional to counting 100.

2.8 Histological investigations 

Histological examinations were carried out after 7 days of exposure to 8 and 16 µL/L RIPEX. The digestive glands and ovotestis were dissected out and immediately fixed in Bouin᾿s fluid. After 1  day of fixation, samples were then dehydrated through a series of alcohols and cleared in xylene. Paraffin wax blocks were made [42]. Ten Sects.  (5–7  μm thickness) were cut and stained with hematoxylin and eosin (Mayer’s H and E). Staining was followed by a good wash with tap water. Histological sections were photographed using a photo-automated camera (Optika, Italy).

2.9 Statistical analysis 

Data were expressed as the mean±standard deviation and analyzed by statistical software of social sciences (SPSS; IBM Corp. Armonk, NY, USA). Two-way ANOVA analysis of variance was used to identify differences between the control and exposed groups, between the diferent concentrations and the time points of exposure. The level of significance was set at p ≤ 0.05.

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Hoda H. Abdel‑Azeem1*, Azza H. Mohamed1 and Mohamed R. Habib2