International Journal of Drug Development and Research

Keywords

Zebrafish, animal model, drug toxicity, drug screening

INTRODUCTION

The introduction of chemicals into the environment by human activities can represent a serious risk to environmental and human health. Therefore, current legislation in European and other industrial countries requires appropriate data on risk assessment for the registration of chemicals, pesticides, biocides and pharmaceuticals (EC 1907/2006). These data include information on the toxicity at various trophic levels and require testing with vertebrates, in particular with fish. Due to the new European Chemical Policy REACH (registration, evaluation and authorisation of chemicals), a dramatic increase in the number of animal experiments is expected [1, 2]. There is a great public demand for the replacement of animal tests for ethical reasons, but industry is interested in alternative testing methods that are less cost intensive and less time and space consuming [3]. Due to these demand we will focus on one of the best described and most popular vertebrate model species the zebrafish (Danio rerio) in developmental genetics and ecotoxicology. Zebrafish is a small vertebrate that can be kept in captivity in large numbers easily [4]. Its generation time is short and most importantly a single spawning can produce hundreds of offspring [5]. Furthermore, zebrafish can be subjected to chemical mutagens and thus many mutants can be produced quickly [6, 7]. Several genes discovered in this species are evolutionarily conserved and have homologs exists between the human and zebrafish genomes (approximately 75% similarity) [8]. The present group of studies investigate bioavailability of different drugs in adult zebrafish and zebrafish fry.

Materials and Methods

Chemicals and solvents

The chemicals, solvents and drugs were of analytical grade and were purchased from Hi-Media Laboratories Pvt. Ltd (Mumbai, India) and Merck Chemicals Company, MO, USA.

Extraction of active pharmaceutical ingredients or pure drugs from tablets

The drugs were extracted by grinding the over the counter drugs with 10ml of methanol: water (3:7) solvents and later on centrifuged at 3000rpm for 10 minutes. The supernatants were collected, transferred into a petriplates and left for drying at 37°C overnight. The crystallized drugs were used for the experimental studies

Animals and maintenance

Zebrafish of uniform size of length (2.6 ± 0.2cm) and weight (1.15 ± 0.1g) were segregated from the stock and acclimatize for 10 days to lab conditions, temperature (27±2ºC), pH (7.5-7.8) and almost normal photoperiod (12:12-h L/D). The fish were divided into eight groups of six each. Control fish (CON); Control fish received control diet throughout experimental period. Group 1-8: Experimental group fish received control diet along with different recrystalized drugs (Table1). Simultaneously fish fry were divided into eight groups and treated with similar recrystalized drugs. The experimental zebrafish were sacrificed at 24hrs and 48hrs by decapitation. Blood was collected and the liver were excised immediately, processed and used for analysis.

TLC separation of experimental zebrafish

About 10μl of the adult zebrafish liver extract and blood sample were spotted on TLC plates and the chromatogram was developed using chloroform: methanol: ammonia solvent in ratio of 6:3:4. The slides were observed under UV transilluminater and the spots were identified. In parallel homogenised fish fry were spotted on the TLC plates and the chromatogram was developed.

Spectral analysis of TLC separated compounds

The TLC separated spots of blood and liver extracts were scraped and dissolved in 30% methanol and centrifuged at 4000rpm for 5min. The supernatants were taken and their absorbances were recorded at 700-200nm.

Results and Discussion

Re-crystallised standard drug

The drugs were recrystalised with solvent and crystals were observed under light microscopy (Fig. 1) to prove that extracted drugs were 100% purity. This was also cross verified with spectral analysis.

TLC separation of experimental zebrafish extracts

Whole zebrafish fry and adult zebrafish blood and liver extract were separated using TLC. The TLC plates were analysed using ImageJ software, which was suggested by the National Institutes of Health (NIH). The fluorescence was obtained in arbitrary units (a.u) which can relatively quantify the amount of drug present. The presence of drug in blood and liver extract are listed (Table 2).

CONCLUSION

The zebrafish is rapidly gaining acceptance as a promising animal model for drug and chemical toxicology [12, 13]. Zebrafish facilitate large costeffective studies that can be combined with a battery of advanced molecular tools for genetic modulation and powerful in vivo molecular visuals readouts to carry out forward genetic or chemical screens in a more rapid and efficient manner than possible with traditional mammalian models [17]. In conclusion, the findings of the current study illustrate 80% similarity of bioavailability of compound in adult zebrafish and fish fry because 7 out of 8 groups were comparable. With the above findings we recommend that the fish fry can be a substitute to adult zebrafish and will be the right model for bioavailability testing due to the fact that the fish fry embryogenesis is rapid and the entire body plan established by 24 hour post fertilization (hpf) and the most of the internal organ like heart, liver and, intestine totally developed by 96 hpf. In addition fish fry are transparent which means organ cells and tissue visualize in vivo and investigated in real-time.

Acknowledgement

The authors are thankful to the Management Committee of The Madras Medical Mission for providing the facilities to carry out the research work

Tables at a glance

Table 1 Table 2

Figures at a glance

Figure 1

Graph 1

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