An Investigation of Polymer Coated Metallic Nanoparticles Mediated Influence on Gut Microbiota of Zebra Fish (Danio rerio)

 

S. Karthick Raja Namasivayam, V. Poornima

Department of Biotechnology, Sathyabama University, Chennai 119, Tamil Nadu, India

 

ABSTRACT:

Nanotechnology is currently gained increased attention in various fields of science and technology like imaging, sensing, targeted drug delivery, gene delivery systems and artificial implants which is increasing the commercial usage of products including drugs in the various parts of the world. Because of their extensive consumption, it is necessary to study the harmful effects of nanomaterials particularly their ecotoxic effect and cytoxicity of nontarget organism (NTOs). In the present study, an attempt has been made to study toxic effect of silver and gold nanoparticles coated with chitosan on the gut microbiota of adult zebra fish adopting culture dependent methods. Respective metallic nanoparticles were synthesized by chemical reduction and stabilization of precursor salt and the synthesized polymer coated nanoparticles were characterized by electron microscopy studies and fourier transform spectroscopic studies. Adults of zebra fishes were administrated with lethal dose 50 (LD₅₀) of polymer coated respective nanoparticles and their stress on gut microbiota studied by analysis of gut tissue homogenate of tested fishes adopting culture dependent methods. Chitosan coated silver nanoparticles brought about maximum reduction of microbial count which reveals drastic reduction of microbial count as increasing time periods. Less toxic effect on microbial count has been inferred from chitosan coated gold nanoparticles.

 

 

 

 

INTRODUCTION:

Due to the physical and ethical issues associated with performing experiments on humans, biomedical research utilizes primarily animal models to study biologic processes conserved between humans and lower vertebrates such as rats and mice. Although these models have significant advantages, they are also expensive to maintain, difficult to manipulate embryonically, and limited for large-scale genetic studies¹.As the most numerous and phylogenetically diverse group of vertebrates, fish teach us important principles about fundamental processes in vertebrate evolution, development and disease processes². Fish have served as useful sentinels to detect environmental hazards and as efficient, cost effective model systems for mechanistic toxicology and risk assessment for many decades³.

 

During the past decade zebra fish have gained a pivotal place in embryology and developmental genetics. Zebra fish and other aquarium fish species have distinct advantages as models for biomedical research including much lower husbandry costs than mammals⁴.

 

The zebrafish, Danio rerio, has emerged as a popular vertebrate model in different areas of research such as developmental biology, genetics, pharmacology, and toxicology in recent years^5,6. Due to transparent embryos and rapid organogenesis, zebrafish were recognized as a tool for developmental biologists in the 1970s. In the 1990s, zebrafish were used for the first vertebrate large scale mutagenesis screen, yielding thousands of mutations, some of which recapitulated human diseases. Several characteristics make zebrafish a convincing tool for drug discovery^7,8. The zebrafish has several features that make it attractive as an animal model. As an oviparous species, zebrafish have external fertilization and development, which allow for easy detection of morphological alterations and manipulation of the transparent embryos⁹. Zebrafish has rapid growth (with all major organs formed within 2-4 days), high egg yield, and a short generation time. Their genetics and developmental biology have been well documented. The sequenced zebrafish genome is being assembled and annotated, and many molecular techniques have been developed to study gene function in zebrafish, including the production of transgenic and mutant fish and the use of transient antisense gene-knockdown methods All these features render the zebrafish embryos/larvae as a convenient platform for pharmacological assessment and screening for potential adverse effects of drugs, particularly with regard to their potential adverse effects during the developmental process^10,11. Recently, zebrafish- based assays have been developed for testing toxicity, including acute toxicity (LC50), organ- specific toxicity and a developmental toxicity ¹². Zebrafish also exhibit similar responses to xenobiotics chemicals as mammals, including induction of xenobiotic enzymes and generation of oxidative stress.

 

Currently, nanotechnology is used in the various fields of science and technology. Hence, nanosized organic and inorganic particles are finding increasing attention in medical applications due to their amenability to biological functionalization. Based on enhanced effectiveness, the new age drugs re-nanoparticles of polymers, metals or ceramics, which can combat conditions like cancer and fight human pathogens like bacteria¹³. Increasing numbers of commercial products, from cosmetics to medicine, incorporate manufactured nanomaterials (MNMs) that can be accidentally or incidentally released to the environment¹⁴. Concern over the potentially harmful effects of such nanoparticles has stimulated the advent of nanotoxicology as a unique and significant research discipline. In the present study, toxic effect of free and polymer coated silver and gold nanoparticles has been evaluated against gut microbiota of zebra fish.

 

MATERIALS AND METHODS:

Synthesis of free silver and gold nanoparticles:

Silver and gold nanoparticles were synthesized by chemical reduction of 0.1 M silver nitrate with 0.1 M sodium borohydride and 0.1 M HAucl4 with trisodium citrate respectively Synthesized nanoparticles were characterized by suitable analytical techniques as described in our previous studies¹⁵.

 

Synthesis and characterization of chitosan stabilized nanoparticles:

Chitosan stabilized nanoparticles were synthesized by the method as described in our earlier studies¹⁵ and synthesized polymer stabilized nanoparticles were characterized by suitable techniques.

 

Evaluation of gut microbion:

Fish stocks:

Fish (Danio rerio) were obtained from local market kolathur, Chennai. The fish were maintained in five different tanks of 150 L capacity filled with natural aerated saline water and the water was replaced every 2 days. Each tank was with dimensions 18x12 inches Temperature was maintained at 30–32°C with a natural light–dark cycle. During acclimation period, Zebra fish were fed once a day, 5 g/100g body wt. throughout the experiment. The pH, dissolved oxygen and total alkalinity were in the range of 7.31–7.49, 7.71–8.12 and 31.3–37.13 ppm, respectively. The fish were acclimatized in the laboratory for a week before the experiments were initiated.

 

Determination of LD50:

Acute toxicity can be determined by the calculation of LD50, i.e., the dose that will kill 50% of animals of a particular species.

 

Estimation of the dose range and percentage of mortality:

An approximate LD50 can be initially determined as a pilot study by a so called ‘staircase method’ using a small number of fishes and increasing the doses of the nanoparticle. Six doses can be chosen for determination of LD50 starting from no death to 100% mortality. In our study for estimation of LD50, 6 doses were mixed in water to respective groups of fishes, 10 in each group. The animals were observed for 24 hours and then 48 hours for any toxic symptoms. After 48 hours, the number of deceased fishes was counted in each group and percentage of mortality calculated.

 

Nanoparticles exposure:

Experiments were carried out in glass aquaria. After acclimation, fish were divided into five groups each group containing 10 fish. One of the groups was maintained as a control in natural tap water. The others were exposed to LD 50 of respective nanoparticles per litre of water. The experiment was carried out for 21days. Experimental aquaria were aerated and test media were replaced every day and there was no fish mortality during these exposures.

 

Microbial count determination:

At each sampling time, five fishes per tank were killed by decapitation Fish gut tissues were dissected under aseptic condition at every time intervals. Isolated guts were individually washed several times with fresh phosphate buffered saline to minimize the possible microbial contamination and used for the study. The gut was homogenized with sterile insect ringer’s solution (IRS) in mortar and pestle.  The homogenate was filtered through Whatman filter paper no. 1 and the pH was measured using pH meter (Mic Datal and Co., Chennai).  The filtrate was serially diluted in sterile saline and 0.1 ml of aliquote was plated on nutrient agar (NA), sabouraud agar (SDA). The seeded NA plates were incubated at 37°C for 24 - 40 hours whereas the SDA plates were incubated at 28°C for 7 - 10 days respectively. Microbial colonies appeared after the incubation period was enumerated and the numbers of colony forming units were expressed as dry weight of the gut.

 

Identification of microflora:

Different morphological microbial colonies were selected, subcultured and stored at 4°C on respective agar slants.  Bacterial strains were identified based on morphological, cultural, biochemical characteristics.

 

RESULTS AND DISCUSSION:

In the present study, gut microbiota mediated stress on zebrafish using free and polymer coated metallic nanoparticles has been carried out. Synthesis of free and polymer coated nanoparticles were synthesized by chemical reduction and stabilization of respective metal precursor by suitable reducer and polymer. Respective nanoparticles thus prepared reveals highly stabilized monodispersive particles with the nano range described in our previous studies¹⁵.^.Synthesized nanoparticles thus obtained were lyophilized and the lyophilized preparation was evaluated against gut microbiota of zebra fish. The zebrafish has several features that make it attractive as an animal model. As an oviparous species, zebrafish have external fertilization and development, which allow for easy detection of morphological alterations and manipulation of the transparent embryos⁹. Zebrafish has rapid growth (with all major organs formed within 2-4 days), high egg yield, and a short generation time. In this study, nanoparticles mediated stress on gut microbiota has been studied. Initially different concentration of nanoparticles was tested to determine LD 50. Table 1-4 reveals LD 50 of respective nanoparticles against zebra fish which was used to study the gut microbiota specially aerobic microbiota  which confirmed by changes in bacterial count.

 

Table 1:LD50 for free AgNps

Group	Dose(ug/lit)	Log dose	% Dead	Corrected %
1	5	1	0	2.5
2	10	1.1	12	12
3	25	1.4	23	23
4	50	1.6	43	43
5	75	1.8	65	65
6	100	2	100	97.5

 

Table 2:LD50 for CS-AgNps

Group	Dose(ug/lit)	Log dose	% Dead	Corrected %
1	5	1	0	2.5
2	10	1.2	12	12
3	25	1.5	24	24
4	50	1.7	45	45
5	75	1.8	65	65
6	100	2.1	100	98

Table 3:LD50 for free AuNps

Group	Dose(ug/lit)	Log dose	% Dead	Corrected %
1	5	1	0	0.1
2	10	1.2	1	0.5
3	25	1.5	3.5	7
4	50	1.7	15	15
5	75	1.8	51	51
6	100	2.1	53.2	53.2

 

Table 4:LD50 for CS AuNps

Group	Dose(ug/lit)	Log dose	% Dead	Corrected %
1	5	1	0	0.1
2	10	1 2	1	0.6
3	25	1.5	3.6	7.2
4	50	1.7	15.1	15.1
5	75	1.8	51.1	51.1
6	100	2.1	53.4	53.4

 

Study groups:

Group 1: Control group.

Group 2: Fishes treated with free AgNp

Group 3 Fishes treated with CS AgNp

Group 4 Fishes treated with free AuNp

Group 5. Fishes treated with CS- AuNp

 

Among the various nanoparticles treatment, both free and chitosan stabilized metallic nanoparticles exhibited high level of gut microbial stress which was confirmed by drastic reduction of bacterial count in nanoparticles treatment. The status of the confirmed environment is reflected in the large and varied microbial communities inhabiting the gut. The indigenous gut bacteria is regarded as a valuable metabolic resource to the nutrition of the host by improving the ability to live on sub-optimal diets, improved digestion efficiency, acquisition of digestive enzymes and provision of vitamins¹⁶. The contribution of gut microbiota to the nutrition and disease suppression was also studied by Gut microflora act as a reflection of the environment and incidence of entomopathogens.

 

 

Fig 1.Inhibitory effect (%) of F-AgNps on gut bacterial count at different time periods (hour)

 

Figure 2.Inhibitory effect (%) of CS-AgNps on gut bacterial count at different time periods (hour)

 

Figure 3.Inhibitory effect (%) of F-AuNps on gut bacterial count at different time periods (hour)

 

Figure 4.Inhibitory effect (%) of CS-AuNps on gut bacterial count at different time periods (hour)

 

Inhibitory effect of bacterial count was gradually reduced with respect to the increasing time periods (Fig 1-4).

 

A gradual increase in inhibitory effect was observed from initial exposure time and maximum reduction in increased incubation time. Both free and chitosan stabilized silver nanoparticles shows remarkable reduction in the bacterial count at increased period of exposure.92.0 and 92.5% of inhibitory effect on bacterial count was inferred from free silver and chitosan stabilized nanoparticles treatment during 72 hours of exposure. Among the various nanoparticles, silver has the potential activities like anti bacterial and anti cancer activities. There are many commercially available silver based anti bacterial agents available in the various parts of the world and the various biological mechanism which confirms antibacterial activity. Interestingly, both free gold and polymer stabilized gold nanoparticles shows less inhibitory effect at all the tested time periods. Bacterial count was not affected as in the control during all the tested time periods. Bacterial species that were isolated from gut of control group reveals the presence of Bacillus sp, Pseudomonas sp, Micrococcus sp, Enterobacter sp, Alcaligenes sp Bacterial colonies obtained in plate count were purified and morphological different colonies were selected. Pure cultures were maintained on nutrient agr slant. Identification of the bacterial species was carried out by morphological, cultural and biochemical characteristics which all shows the occurrence of the above mentioned bacterial species. In treatment group of free AgNps and CS-AgNps category, Bacillus sp and Pseudomonas sp were recorded (Table 5).

 

Table 5.Effect of nanoparticles treatment on the bacterial composition

+ present

-Absent

 

No changes in species composition of bacteria were recorded in both free AuNps and CS-AuNps treatment. Further studies based on culture independent methods will helpful to explore bacterial communities associated with gut of zebrafish and their changes to the environmental stress.

 

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Accepted on 30.07.2017          © RJPT All right reserved