Zebrafish in Toxicology Research: Advantages and Limitations

Zebrafish in Toxicology Research: Advantages and Limitations The abuse of psychoactive substances is a serious public health problem and understands the brain disorder induced by psychoactive substances is an important challenge in biomedical research that requires extensive clinical and preclinical investigation (Neelkantan et al. 2013; Stewart et al. 2011). Recreate the complex network of neurochemical interactions in organisms in vitro is not possible, especially for psychotropic drugs (Riehl et al. 2010). In this way, experimental animal models is a important tool for investigation of the toxicology and chemistry of the drugs of abuse. Animal models in toxicological and pharmacological research using mammals present high cost, consumes large amounts of chemical compounds and are laborious to perform the tests and maintenance of animals. In this context, animal models such as the zebrafish, which outweigh these disadvantages, are an interesting and promising alternative in research. Since the 1970s, the zebrafish (Danio rerio) is used in research in neuroscience and developmental biology (Serra et al. 1999). In the 1990’s, zebrafish were used for the first vertebrate large scale mutagenesis screen. In last few years, the use of zebrafish (Danio rerio) in scientific research has increased rapidly in other science fields, including Pharmacology and Toxicology (Chakhaborty et al. 2009). The zebrafish represents an interasting model for integration of research of genetic, neural and behavioral aspects (Miklà ³si and Andrew, 2006). The coupling between behavioral assays and analytical and molecular techniques permits the elucidation of mechanisms of toxicity, the test of new drugs for therapeutic treatments and the study of new drugs (Tierney et al. 2011). The aim of this paper is to outline recent developments and futures perspectives in drugs of abuse research with zebrafish, in Experimental and Analytical Toxicology. Zebrafish as a new animal model in Toxicology: advantages and limitations The zebrafish is a small tropical fish native of northern India and adjacent countries. Many factors and animal characteristics make zebrafish an attractive and efficient model to analyze the mechanisms of action and effects of drugs in general (Chakhaborty et al. 2009). Zebrafish and humans share about 75% of their genome and have physiological similarity (Chakhaborty et al. 2009, Zhu et al. 2014). Genome, transcriptome and proteome of zebrafish have been widely studied and described (Maximino et al. 2010). The similarity level between zebrafish and humans is also observed in the nervous system (Mathur and Guo, 2010). Zebrafish are small (at adulthood, 2.5 – 4.0 cm) and maintenance costs of this fish in laboratory are considerably low, permitting logistical and economical advantages over rodent models (Key and Devine, 2003). The fertility rate and the number of embryos generated are higher those in mammalian models (Chakhaborty et al. 2009). In general, an adult female can produce 200-300 eggs and reproduce 2 or 3 times per week (Blaser and Gerlai, 2006; Gerlai 2003; Patton and Zon, 2001; Zon and Peterson, 2005). The zebrafish cycle of life and development period is well-characterized and short (3 to 4 months) (Cadet, 2009; Hill et al. 2005). The body is formed in the first 24 hours post fertilization (hpf) and the internal organs are fully developed at 96 hpf (Chakhaborty et al. 2009; De Esch et al. 2012b; Ninkovic and Bally-Cuif, 2006b; Parng et al. 2002; Patton and Zon, 2001). The development outside of the uterus and the optical transparency of eggs and tissues during embryogenesis allows the visualization of tissues and organs in vivo, making possible the visual analyses in real time of early developmental processes, organ morphology and dysfunctions caused by drugs of abuse and quantification of cell proliferation and cell death in specific tissues (Chakhaborty et al. 2009; Friedrich et al. 2010; Hill et al. 2002; Ingham 2009; Miklà ³si and Andrew, 2006;; Mathur and Guo, 2010; Ninkovic et al. 2006b; Parng et al. 2002; Patton and Zon, 2001; Peterson and MacRae, 2012; Xu et al. 2011). In addition, blastomeres of zebrafish are large and stable for biophysical and electrophysiological assays (Zhu et al. 2014). The zebrafish has become a widely utilized model organism in pharmacological and toxicological research, particularly due to evidence that they may share with humans and other mammals some key receptors targeted by drugs of abuse (Miller et al. 2014). In addition, zebrafish are highly social animals which enables them to display robust behavioral responses, such as shoaling, aggression and social preference, and emerge as a sensitive alternative model to investigation of drugs of abuse-evoked states (Cachat et al. 2013; Pham et al. 2012). Another advantage is that the size of zebrafish provides a rapid absorption of drugs via the gills and leads to bioaccumulation in Central Nervous System (CNS) and other tissues (Echevarria et al. 2008). Zebrafish is amenable to molecular and genetic analysis (Bailey et al. 2013; Chakhaborty et al. 2009; Miklà ³si and Andrew, 2006; Parng et al. 2002). Since genetic mutations can interfere in brain function, the use of molecular and biochemical techniques in zebrafish allows the identification of molecular substrates for drugs in brain and the analysis of function and regulation of the genes, the production of transgenic strains and the induction of specific mutations, inducing overexpressing of the genes or decreasing genes expression (Cadet 2009; Goldsmith 2004; Key and Devine, 2003). Use of zebrafish mutant strains has allowed the understanding of mechanisms and pathways and neural expression of specific genes. Due to the increasing progress in this area, studies of mutant zebrafish have investigated specific behaviors, diseases, deformities and functional processes (Spitsbergen and Kent, 2003). The performance of point mutations in zebrafish, generating mutant lines, enables the molecular investigation of the mechanism of action of drugs of abuse, determining specific receptors and target molecules. Although the model advantages are numerous, some limitations should be considered in the use of zebrafish in toxicological, pharmacological and neuroscience research. The normal or defective functioning of the human brain is complex to be fully modeled in zebrafish brain. Some areas do not have the same degree of development of the mammalian brain, making it difficult to map for mammalian counterparts (Kalueff et al. 2014; Rinkwitz et al. 2011; Stewart et al. 2014). Moreover, the social behavior is not clearly defined in fish larvae (Kalueff et al. 2014; Stewart et al. 2014). Zebrafish has a genome 30-40% tetraploid due to an genome duplication with some of their genes having two copies instead of one as in mammals – and one of these two genes are not represented in the human genome (Alestrà ¶m et al. 2006; Klee et al. 2011; Stewart et al. 2014). One disadvantage associated with the zebrafish model is the fact that some drugs are not soluble in water, making it difficult to administration by immersion. This potential problem can be solved by using organic solvents or other routes of administration (Stewart et al. 2014). In other hand, exposition to drug by immersion reduces the stress-induced by injection (Stewart et al. 2011b). Finally, although the barrier between brain and blood presents development similar to humans, the distribution of some drugs in organism may be different in different species (Stewart et al. 2014). Although the pattern has some limitations, the advantages over conventional models mammals make zebrafish a very interesting alternative in research. In this sense, the use of zebrafish in research involving drugs of abuse, in order to characterize drugs of abuse effects and to quantify these compounds in a biological fluid from the animal is increasing and represents a great target for research. Zebrafish research to study the drugs of abuse The conduct of toxicology research in laboratory animals has become a well-established and essential practice and chemical and toxicological information on chemical compounds and drugs are obtained from the results of these studies (Gad 2007; Olson et al. 2000). The use of animals in research has several advantages: low cost, easily use and functional homology with humans (Gerlai et al. 2010).The animal experiments to predict the action, metabolism and effects of drugs of abuse in humans are extremely important in Toxicology. Introduced as a model for neural development by George Streisinger in the 1960’s, zebrafish has become a promising aquatic model for study of drugs. The zebrafish is an efficient alternative model of drug delivery via the gills, by direct application of drugs in water and posterior submersion of the animal. Between 12 and 14 days after fertilization (dpf), molecular oxygen is mainly absorbed by the skin, suggesting that this is the main route of uptake for small molecules, and after 72 h the embryos begin to swallow indicating the availability of the oral route for absorption of chemical compounds (Goldsmith 2004). Exogenous compounds, such as ethanol, have been shown to rapidly enter in systemic circulation of the fish, demonstrating the high sensitivity of the zebrafish to various psychotropic agents (Dlugos and Rabin, 2003; Echevarria et al 2006; Kyzar et al. 2012). In the larval and embryonic stage, the performance of rapid and high-throughput analysis of multiple behavior s and screening of chemical compounds is possible and suitable (De Esch et al. 2012b; Guo 2009; Richendrfer et al. 2012). The first response to the action of a psychoactive substance is the behavior. In this sense, the zebrafish has been widely used in the development and validation of behavioral assays to different drugs of abuse, due to its sensitivity to drugs and their robust responses. Locomotor activity, learning, sleep, aggression, social and antipredator behavior are some of the observed and well-characterized behavioral parameters in zebrafish (Bailey et al. 2013;Cachat et al. 2013; Guo 2004; Pham et al. 2012; Spitsbergen and Kent, 2003). Endocrine responses to stress in zebrafish are also an important tool for toxicological effects induced by drugs of abuse. In zebrafish, the hypothalamus-pituitary-interrenal axis (HPI) is homologous to the hypothalamic-pituitary-adrenal axis (HPA), found in mammals and humans, and cortisol is involved in physiological responses to stress situations (Stewart et al. 2010). Another interesting pattern of physiological change for toxicological investigation of drug in zebrafish is the color of the skin. As mammals, fish have cells containing pigments in the collagen layer of the dermis and these cells have hormonal and nervous regulation (Nguyen et al. 2013). In this sense, effects on skin color may contribute in part of the elucidation of the mechanism of action of many drugs of abuse. The toxicity induced by abuse of drugs in the digestive and cardiovascular systems of zebrafish can also be determined. These systems are developed and become fully functional in the first days after fertilization (Patton and Zon, 2001). The characterization and monitoring of blood vessels in embryos is easily achievable (Rubinstein 2003). The heart rate is an indicator of cardiac toxicity widely used. Morphological examination of the heart and digestive organs can be performed to identify organ-specific toxicity of drugs of abuse (Rubinstein 2006). For example, the effects of environmental toxin tetrachlorodibenzo-para-dioxin (TCDD) on heart rate (Henry et al. 1997), the functional lipid metabolism (Farber et al. 2001), the effects of fungicide riphenyltin acetate (APTT) in hepatocytes (Strmac and Braunbeck, 1999) were investigated using histological and immunochemical analysis. The development of analytical techniques to determine compounds of interest in biological fluids zebrafish also plays an important role in the elucidation of mechanisms of action and toxicity of drugs of abuse. Moreover, it is extremely important to develop methods of extraction and concentration of analytes and biomarkers that make possible dosages required to understanding toxicological aspects of these drugs of abuse. The main analytical methods developed to study the drugs of abuse in zebrafish are listed in Table 1. The new psychoactive substances (NPS) that have emerged in recent years have action and effect mechanisms partially or totally unknown. According to UNODC, the number of NPS significantly increased between 2009 and 2013 (UNODC 2014). In many poisoning deaths caused by these unknown substances, the lack of information makes it difficult to identify the cause of death. Therefore, the use of zebrafish in research represents an important tool to scan and evaluate the chemical and toxicological properties of both known and unknown drugs of abuse. Marketing Design and Innovation: Wrigley Marketing Design and Innovation: Wrigley Wrigley is a well-known global brand dealing in wide range of confectionery products such as chewing gums, chewy and hard candies, mints, and lollipops. It is operating in nearly 50 countries and distributing its products in more than 180 countries. The company is based in Chicago, Illinois and was founded in 1891. Some of its brands i.e. Juicy Fruit, Wrigleys Spearmint and Altoids have heritage of more than hundred years. Wrigley is famous for its innovative products that are introduced into its product line from time to time. Wrigley uses best ingredients and packaging materials for its products and the core principles on which it operates include quality, freedom, efficiency, responsibility and mutuality (Wrigley.com, 2017). Late in 2014, Wrigley merged its Orbit and Extra brand but it launched Extra with new improved taste in 2015. As per the new market trends for health and taste, Extra is a new sugar-free gum available in single packs, multipacks and bottles introduced in more than five different tastes. The product is launched for Australian and New Zealand market. One of the best attributes of Wrigleys product is innovation and the concern for consumers health. Wrigley continuously update the flavors of its Extra brand to please the palate of its valued customers. UK confectionery market have been observed to have a high volume consumption and it stands as the sixth largest in terms of confectionery consumption. The confectionery market is driven by pleasure, convenience, drug replacement, snacking and innovation in products (Globaldata, 2016). Consumers are becoming health conscious due to the increasing issues like diabetes and obesity. Dentists are also increasing the awareness among people about oral health and the tooth related diseases such as plaque and saliva generation etc. dentists are making the consumers aware of the benefits of chewing. Due to the increased health consciousness, people are likely to take more products for treatment and prevention of such issues. Wrigley has launched Extra and Extra Active in association with The Australian Dental Association (ADA), New Zealand Dental Association (NZDA) and the FDI World Dental Federation. According to Country Report Wrigleys Extra marked the growth in 2016 because of its sugar-free and oral care features (Country Report, 2016). Now, Extra is involved in cause-related marketing and is making associations with the dental professional to educate the families about the significance of oral health. This brand contributed about one third of the percentage of total value shares of the Wrigley Company and is expected to continue to gain highest share because of its uncountable benefits (Country Report, 2016). Maslow Hierarchy of Needs theory   Ã‚   According to the Maslow hierarchy of needs theory, Wrigleys product Extra attempts to satiate the physiological needs of an individual. Although its not basic necessity for life but oral health and refreshment are necessary for an individual and Extra` is capable of meeting these needs. It also satisfies the social needs of an individual because it boosts the confidence and reduces pressure and anxiety according to the research conducted by psychologists (Smith, 2012). Refreshing breath and sweet smell is necessary if an individual is attending a social gathering or event which is satiated by Extra i.e. to meet the love and belonging needs. People with high level of income usually buy Wrigleys Extra which also satisfy esteem needs because it represents the status quo of the individuals consuming this brand. The self-actualization needs of an individual can be satiated by Extra because it gives a new touch to the lifestyle of an individual i.e. a refreshing breath and increased confid ence. Diffusion of Innovation Model Diffusion is defined as the process by which an innovation product is passed on to the members of a social system with the help of different channels over a specified time period. There are five types of people involved in the diffusion of innovation process i.e. innovators who are adventurous and are ready to the test the product as soon as possible; early adopters adopt the idea and are aware of the benefits associated with change; early majority adopts the idea before an average person does; late majority adopts the idea once a greater majority has tested it, and laggards are cynical to change and do not adopt the idea usually (Smith and Song, 2004). Due to increased awareness among consumers about oral health, the percentage of people using Extra brand is increasing. Wrigleys Extra has been rated 8.9 out of 10 by the consumers which means that they are early adopters who have adopted the product within a time span of two to three years (infoscout.co, 2017). The other reason for product adaptability is the range of tastes available i.e. the consumers consume different tastes of the products to please their palates. Consumers prefer products which are healthy and Extra is healthy in terms of oral care and its sugar-free as well. Extra is clinically proved by the professional dental associations and it also stimulates the saliva by 10 times. It is very important to identify the needs of consumers and become aware of the market trends before launching a new product. Because the consumers are becoming increasingly health conscious, Wrigley has identified recent trends in the market and launched Extra into the market. Kano model Customers needs change over the period of time due to the changing market trends and in the recent business environment, customer is considered as the king of a business. According to the Kano model, a product must be developed considering the basic needs of customers, performance needs and excitement needs i.e. basic needs are hygiene needs while excitement needs are delighting needs and these delight needs later one become basic needs and then customers need something new (Wang and Ji, 2010). Wrigleys Extra is a product that can satisfy the fun and pleasure needs along with the health needs of an individual. The hygiene needs of Wrigleys Extra are the promotion of oral health, prevention of teeth caries, and plaque reduction etc. while the fun and pleasure attributes of Extra satisfy the delight needs. With the innovation of brand these delight needs will become the basic needs of an individual. The bottom line is that the product should have the attractive and must-be attributes that can fulfill the needs of customers for which they are using the product. These attributes of Extra are health and bacteria prevention ingredients. Product anatomy During the product development, the anatomy of product is important to analyze in terms of the benefit it will offer (Blythe, 2017). The product anatomy is conducted to match the consumer trends with the product offered by a particular brand. The core product of Wrigleys Extra is the oral health and its sugar free i.e. cleans food debris, neutralizes plaque acids, strengthens teeth, reduces dental caries, prevents tooth decay, and stains. The generic product of Wrigleys Extra is that its available in wide range of flavors i.e. strawberry, peppermint, spearmint, white lemon lime, bubble mint, water melon, berry and orange cream etc. The expected product is that it stimulates saliva and refreshes individuals breath. And the augmented product of Wrigleys Extra is that it is available in different types of packaging i.e. 14 pieces and 10 pieces single pack, 28 and 42 pieces multipack, and 46 pieces bottle (Desfosse, 2017). This product has delighted the palate of numerous customers which is reflected in the increased sales figure of Wrigley. A report showed that one third of Wrigleys sales of 2016 were contributed by Extra (Country Report, 2016). It shows that people are continuously using the Wrigleys Extra for their oral health. The development of Wrigleys Extra has shaped the overall gum market and has proved to be a market leader in 2016 by increasing the overall sales of Wrigley. The prime cause for increased value of Extra is its association with the professional dental associations and being a sugar-free chewing gum. The brand awareness is created by educating the families and general public through the use of creative and educative advertisements (Schultz, 2015). According to statistics of 2015, the sugarless and breath fresheners has seen considerable increase in dollar and unit terms. The sales of Wrigleys Extra increased to 9.3% from 8.6% a year ago. This is because consumers are adopting the more socially acceptable mint and refreshment gums for refreshing themselves. The sales of Wrigley Extra are expected to grow in the near future because the dental care specialists are positioning the oral care and sugar free gums as more important for the consumers to chew after and between the meals (CSP Daily News, 2015). It will be easy for Wrigley to retain its customers for Extra because of the cause-related marketing and its association with the professional dental associations. Due to the increased consciousness and awareness among the people about obesity, they are reducing more and more on the snacks and this is the best alternative that can be replaced for snacks. This trend of marketing is likely to increase the sales figure for Extra chew ing gum. Another important marketing milestone of Extra is that its associated with the Action for Children charity donations from sales of Extra packs. Balanced scorecard Business scorecard is the metric used for the purpose of aligning different strategies with the vision and mission of an organization. It undertakes the newly formed strategy of a particular business and transforms it into marching order on daily basis. A newly developed product can be seen from four perspectives according to the balanced scorecard. These four perspectives are learning and growth perspective, business process perspective, customer perspective and financial perspective (Violet and Josiah, 2011). Learning and growth perspective: According to the recent statistics for Extra gum, it can be evaluated that the consumers percentage using this product is increased a certain extent during 2016 and is expected to grow further (Country Report, 2016). Although Extra gum was once merged with the Orbit brand of Wrigley but later on it was relaunched with improved taste and packaging that attracted a whole new lot of consumers and increased the market share by 71%. Customer are satisfied with Extra and Extra Active brand of Wrigley due to its string oral care positioning and sugar-free attribute. Business process perspective: Extra gum was launched with new taste and packaging and it was brought back to the market due to its increased demand from the customers. Management has launched the product after conducting complete market research and clinical importance of the product. It is made with best possible ingredients i.e. real sugar and no artificial colors are added into it. The unit sales of Extra gum are increased from 8.6% to 9.3% that shows the efficient procurement and distribution capabilities of Wrigleys Extra (CSP Daily News, 2015). Customer perspective: Extra is available in different tastes, different packets size and is suitable for customers of any age. The product is best for chewing before and after the meals and is mainly targeting the oral care of children and aged people. It is the best replacement for the customers who want to quit smoking and it can also be used after smoking. Customers are satisfied about this product which is evident from the increased sales figure and the reviews of customers for this product (Wrigley.com, 2017). Financial perspective: Extra gum has contributed one third of the total sales of Wrigleys overall sales figure. The Country Report (2016) quotes that The company is expected to edge up by around a third of a percentage point overall to reach close to 89% value share. The companys share gain in 2016 will be solely due to its Extra brand, which alone will gain over a percentage point in the year and dominate sales with over 71%. It means that the brand is also performing well in terms of finance. The overall position and performance of Extra gum suggest that due to the oral care positioning and cause related production and marketing of the product, Extra will be put as an important element for the oral care regimes. The competitive advantage of the product is that it is available in different sizes, flavors, packets, is nutritional and perfect for all age users and is available in more than 180 countries. Extra gum is one of the best products designed by Wrigley for oral care and is sugar-free which was once kicked out of the market due to the other brands offering same product. Based on the heritage of more than hundred years and the best quality, Extra reclaimed its leadership and innovative place in the market after its launch with new tastes and packaging. The innovation and leadership attributes of Wrigley are renowned in the market. The company should strive its best and keep an eye on the market to continuously upgrade the brand in terms of packaging and tastes so that it may not lose the market share. References Blythe, J. (2017). Product Anatomy. [Online] SAGE Books. Available at: http://dx.doi.org/10.4135/9781446221617.n17 [Accessed 18 Mar. 2017]. CSP Daily News. (2017). Candy: Mints/Gum/Breath Fresheners 2016. [Online] Available at: http://www.cspdailynews.com/category-data/cmh/candy/candy-mintsgumbreath-fresheners-2016 [Accessed 18 Mar. 2017]. Country Report, (2016). Confectionery in the United Kingdom. Country Report. Euromonitor international, p.57. Desfosse, R. (2017). Chewing Sugarless Gum Can Prevent Tooth Decay. [Online] Colgate. Available at: http://www.colgate.com/en/us/oc/oral-health/basics/nutrition-and-oral-health/article/chewing-sugarless-gum-can-prevent-tooth-decay-0213 [Accessed 18 Mar. 2017]. Globaldata, (2016). Consumer and Market Insights: Confectionery in the UK. Market Research report. 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