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Toxicity testing: in vitro models in ecotoxicology
C H A P T E R
34 Toxicity testing: in vitro models in ecotoxicology Justin Scott and Matteo Minghetti Department of Integrative Biology, Oklahoma State University, Stillwater, OK, United States
34.1 Overview of the use of animals in toxicology 34.1.1 Use of animals in scientific research: historic perspective Animals were used by early Greek philosophers and physicians such as Aristotle (384 322 BCE) and Galen (CE 129 210) to advance our understanding of anatomy, physiology, pathology, and toxicology. Since then advancements in these disciplines have relied heavily on animal experimentation. However, while the use of animals in scientific research has undoubtedly contributed to the advancement of biomedical research, it has also raised ethical concerns. The debate on the ethical grounds for animal experimentation is depicted in an iconic painting from Joseph Wright “An Experiment on a Bird in the Air Pump” in 1768.1 The painting depicts a natural philosopher, recreating one of Robert Boyle’s air pump experiments, in which a bird is deprived of air before a group of observers. The group exhibits a variety of reactions from shock to curiosity. What is most relevant about
An Introduction to Interdisciplinary Toxicology DOI: https://doi.org/10.1016/B978-0-12-813602-7.00034-X
the painting is its illustration of the variety of reactions that animal experimentation can induce in the general public. In modern times academic debate, pressure from the general public, and an increase in animal rights’ movements have resulted in government legislation that protects animal welfare. For instance, in the United States, the Institutional Animal Care and Use Committees and in the United Kingdom the Animals (Scientific Procedures) Act were formally instituted in 1986 to review and approve all government funded animal research. Around one hundred million vertebrate animals are used worldwide annually for experimental and regulatory purposes.2 Over 50% of these animals are mice, while fish account for around 10%; a figure that has been increasing over the past 15 years.3 Russell and Burch’s The Principles of Humane Experimental Technique 1959 was pivotal in emphasizing a need for alternative approaches to animal testing and introducing the concept of the “3Rs” (reduction, replacement, and refinement). Over the past three decades, there has been increased interest among the academic, regulatory, and
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private sector communities toward in vitro alternatives to vertebrate animals used for ecotoxicity testing. In vitro alternatives consist of isolated vertebrate cells, biochemical tests, and computer models. A focus exists to move away from, and eliminate altogether, the use of live vertebrate testing specimens due to increasing ethical concerns and the necessity for efficient and less expensive high-throughput testing alternatives. For instance in 2013 the European Commission banned cosmetics, personal care products, and ingredients tested on animals. This process took 20 years of debate between regulators, academia, and industry and was made possible by the development of efficient in vitro alternatives and by sustained public pressure.4,5 Currently most regulatory methods in ecotoxicology still use vertebrate animals (mainly fish) and lethality as a measured endpoint to assess the toxicity of chemicals. Classical acute toxicity testing requires the calculation of Lethal Dose 50% (LD50). The value of LD50 for a substance is the dose required to kill half of a tested population after a specified test duration. Such methods are popular for the ease of execution and interpretation, but do not necessarily generate useful information to predict the substance’s mode of toxic action. Therefore the development of alternatives to whole animal testing, which allow the study of the mechanisms of toxicity in a more ethical and cost-effective manner, is relevant and timely.
34.1.2 Alternatives to animal testing in ecotoxicology Ecotoxicology focuses on preventing, monitoring, and alleviating harmful effects of anthropogenic activities on the environment. Due to the increased number and volume of chemicals developed for societal and industrial progress, pressure on the environment is increasing. A major concern is the negative
impact on the aquatic environment. Contributing sectors are: agriculture, pharmaceutical and chemical production, petrochemical refinement, mining operations, animal farming, power and utility operations, as well as man-made ecological disasters. Environmental protection agencies in most of the western world have developed legislation to routinely monitor and assess the state of the aquatic environment (e.g. Clean Water Act— USA; Water Frame Directive—EU). In addition, specific legislation has been developed to regulate the registration of new chemicals and evaluate potential environmental impacts.6,7 While there are differences in the standards and guidelines across borders, the shared goal to reduce and/or eliminate anthropogenic effects on the environment has also now been extended to reducing the use of vertebrae animals in toxicity testing.6,8 The US Environmental Protection Agency (EPA) has initiated the Toxicity Forecaster (ToxCast) and Aggregated Computational Toxicology Resource, which uses in vitro highthroughput screening approaches and computational toxicology approaches to rank and prioritize chemical toxicity.9 Other organizations, including the Alternatives Research and Development Foundation (ARDF), Interagency Coordinating Committee on the Validation of Alternative Methods, and European Centre for Validation of Alternative Methods, are also pursuing advancements in toxicity testing to identify and validate in vitro alternatives to reduce the number of live organisms for in vivo testing. However, most toxicity testing for the determination of potentially harmful chemicals still relies on the need for live vertebrates. For instance, in the United States alone, three million fish are used every year for Whole Effluent Toxicity (WET) testing.10 Several in vitro alternatives have been developed to determine the ecotoxicity of chemicals; however, the validation of alternative methods remains critical to allow the use of such
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34.2 Alternative methods in regulatory ecotoxicology
in vitro alternatives in regulatory toxicology. Specifically, there is a need to provide regulatory agencies, whose focus is to enforce methods aimed at preventing and monitoring harmful anthropogenic activities, with reliable and reproducible results. The aim of this chapter is to describe the current state of in vitro alternatives highlighting their strengths and weaknesses and suggest future directions to validate the most promising alternative methods in regulatory ecotoxicity testing.
34.2 Alternative methods in regulatory ecotoxicology In vitro alternative methods approved for regulatory purposes include the fish embryo test (FET), approved by the Organisation for Economic Co-operation and Development (OECD) and by the International Organization for Standardization (ISO) (OECD 236; ISO 1508811,12) and the fish gill cell line (RTgill-W1) approved by ISO (ISO 21115-201913). The OECD approval process of the FET took nearly a decade and involved two phases. Phase 1 aimed to evaluate the transferability and the intra- and interlaboratory reproducibility of the FET after exposure to seven chemicals. Phase 2 built on this by further testing reproducibility with the addition of 13 chemicals and covering specific areas of use (i.e., chemicals, pharmaceuticals, pesticides, and biocides).14 This process resulted in the approval of the use of the FET to replace the in vivo acute toxicity test (OECD Guideline 20315) for regulatory testing.16 The ISO standard 15088 has also been developed as a substitute for the acute fish toxicity test (ISO 7346-1 and 7346-2) when applied to waste water. Additionally the use of cytotoxicity assays using fish cell lines (see Section 34.2.2.2) has been proposed as a viable alternative to the fish acute toxicity test.17 19 The recent determination of the rainbow trout
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(Oncorhynchus mykiss) gill cell line, RTgill-W1, as an in vitro alternative to the fish acute toxicity test resulted in the approval of the method ISO 21115-201913. Moreover, the demonstration of inter- and intra-laboratory reproducibility of the RTgill-W1 cell assay19 places this method a step closer to OECD approval. Mathematical models and the use of invertebrates have also been used as valid alternatives to vertebrates in regulatory toxicology. Quantitative structure activity relationship models (QSAR) are mathematical models used by European and North American legislations to predict both bioaccumulation and short- and long-term toxicity in fish. QSAR are nontesting models used to predict toxicity of chemicals based on their physical and chemical properties and on the principle that similar substances have similar biological activity. QSAR predictions for acute toxicity and bioconcentration in fish are commonly used and well accepted for regulatory purposes (OECD 17120). Additionally among the alternative methods under investigation, the use of invertebrates, such as the Hydra21 and the Daphnia,22,23 can effectively detect the toxicity of chemicals dissolved in water. However, the simplicity of these organisms, while being a methodological strength, is also a limitation, since their physiology is much less complex than that of vertebrate animals. Nonetheless, they represent good alternative models for preliminary toxicity screening, which can substantially reduce the use of vertebrate animals. Tiered testing approaches for chemical registration, risk assessment, compliance with exposure levels, and fate of contaminants all use aquatic toxicity testing components with both vertebrate and invertebrate in vivo models. The following alternative model systems have been chosen for their use and significance in regulatory ecotoxicology and for their potential development into an approved alternative approach to whole vertebrate animal testing.
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FIGURE 34.1 Panel A illustrates the zebrafish (Danio rerio), the fathead minnow (Pimephales promelas), and the African clawed frog (Xenopus laevis) developmental stages relevant for embryo toxicity and teratogenicity assays. According to the Organisation for Economic Co-operation and Development, fish and amphibian embryos/larvae are considered nonprotected until the animal is capable of independent feeding, occurring approximately at 120, .176, and 96 h postfertilization for the zebrafish, fathead minnow, and Xenopus, respectively. Panel B illustrates the measured toxicological endpoints for the zebrafish fish embryo test (i.e., coagulation of the embryos; lack of somite formation; nondetachment of the tail; lack of heartbeat), which are measured at 24 h intervals until test termination.
34.2.1 Fish and amphibian embryos The FET and the Frog Embryo Teratogenesis Assay-Xenopaus (FETAX) can be defined as alternatives to animal tests based on a convention that establishes that embryos are not protected animals up to a certain stage of development and can serve as a viable in vitro alternative. The OECD’s concept of a protected animal is defined as any living vertebrate, other than man, at the point in which it becomes capable of independent feeding. Therefore organisms at embryonic and eleuthero-embryonic stages, where the organism is still using the yolk sac as
energy source, are not protected animals. The US Office of Laboratory Animal Welfare sets a more stringent definition for fish and amphibians and considers hatching as the point at which organisms are protected.24 Fig. 34.1 shows and compares fish and amphibian embryo life cycles with their respective method for ecotoxicology. The FET has gained promising strides both in Europe with the use of zebrafish FET as an alternative to acute fish testing, and in the United States with the EPA’s use of fathead minnows’ larval survival and teratogenicity tests and chronic toxicity.25 The FET is also utilized for several regulatory
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FIGURE 34.2 Preparation of gill cell cultures from rainbow trout (Oncorhynchus mykiss). (A C) Gill arches are excised, gill filaments are separated and washed in phosphate buffer. (D) Gill cells are washed repeatedly with antibiotic and antifungal solutions and dissociated from each other using a cell strainer and enzymatic digestion (i.e., trypsin digestion). (E) Cell viability and total numbers are evaluated using the trypan blue exclusion assay with a hemocytometer or automatic cell counter. (F) The primary gill cell culture system is generated by seeding two primary gill cells cultures (steps A E) on a polyethylene terephthalate porous membranes in two consequent days. (G) Double-seeded primary gill cultures form a tight epithelium when cultured in symmetrical conditions for 5 7 days and when a trans epithelial electrical resistance of above 5000 Ω cm2 is reached cells can be maintained in asymmetric conditions and tolerate direct exposure to fresh water. (H) A gill cell line was isolated using a similar protocol (steps A E). Primary gill cells were cultured as monolayers on culture flasks and passaged several times until the RTgill-W1 cell line was isolated. (I) RTgill-W1 cells can be cryopreserved and thawed when needed for cytotoxicity assays and other applications.
biomonitoring protocols including the International Maritime Dangerous Goods Code, Federal Insecticide Fungicide and Rodenticide Act, and WET testing methods to detect chronic effects. The FET allows toxicological observations to be made on the organism at the earliest stages of development. Embryos are gathered postfertilization from tanks holding spawning adult breeders and then exposed to aquatic samples to help measure and determine toxicological impacts. Viability of each embryo is visually observed with a microscope at
24 hours intervals over the test duration (acute at 48 and 96 hours postfertilization) to measure the toxicity markers which include: (1) coagulation of the embryos; (2) lack of somite development; (3) nondetachment of the tail, and; (4) lack of heartbeat which is used as endpoint of mortality and also to generate Lethal Concentrations 50% (LC50) values. The FETAX is a method similar to FETs and developed using the African clawed frog (Xenopus laevis) from the amphibian metamorphosis test. The FETAX assay allows monitoring of lethal and sublethal endpoints through a more
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cost effective and expedited acute assay using in vitro approaches.26 Using fertilized frog embryos, three endpoints are evaluated (mortality, malformations, and embryonic growth). Similarly to the FET assay, observations are recorded at 48, 72, and 96 hours, and lack of heartbeat is used to generate LC50 values.27 FETAX studies have been used to assess and predict both ecological and human health hazard evaluations.28 From an ethical point of view, the FET and FETAX do not truly take on the concept of live animal toxicity testing replacement, but rather significantly reduce the number of animals used and refine an approach to measure additional toxicological endpoints. In addition, the idea of fish embryo and larvae welfare has been considered when used in toxicity testing. For instance it has been found that 120 hours postfertilization fish larvae responded to noxious stimuli and that the response was relieved with pain reducing drugs.29 Moreover although FET and FETAX have shown to be effective predictive methods of ecotoxicity of chemicals, they require husbandry practices of breeding adult vertebrate test specimens which have ethical and cost implications.
34.2.2 Use of isolated fish cells 34.2.2.1 Primary cultures Primary cell cultures are prepared by excising a selected tissue and by maintaining the isolated tissue cells in vitro. Primary cells retain most of the tissue cellular physiology but remain viable for a relatively short time, from a few hours to a few weeks. Cell viability should be monitored to determine the extent of time primary cells can be used for experiments. For instance, intestinal cells isolated from rainbow trout (O. mykiss) were shown to remain viable for 4 hours using the trypan blue exclusion assay.30 On the other hand, primary gill cells cultured on permeable membrane
remained viable for at least 2 weeks and primary hepatocytes prepared to form spheroid structures remained viable for up to 4 weeks.31,32 Culturing techniques designed to maintain the in vivo cellular architecture and microenvironment, such as culturing cells on porous membranes or allowing the formation of 3D structures (e.g., spheroids), have also been shown to allow better retention of the in vivo tissue-specific features in comparison to cell monolayers (i.e., 2D cultures). Primary gill cells cultured on polyethylene terephthalate (PET) porous membranes retain several of the features of the gill epithelium in vivo, including a high trans epithelial electrical resistance (over 20 kΩ cm2), expression of apical tight junction protein and presence of mitochondria rich cells which are key feature of this tissue.31,33 In fish the gill is extremely sensitive to environmental insults and serves as a multifunctional organ. Considering its large surface area, it is the major site of toxicant exposure and uptake.34 The cost associated with gill failure is inevitably the death of the organism. Thus the gill represents an ideal model to be used as an alternative to acute fish toxicity testing. Primary gill cells have been used for fish physiology and toxicology studies including biomonitoring of pollutants in river waters.31,35,36 Moreover with the 3Rs in mind, the use of primary gill cells reduces the number of fish used for acute toxicity tests, as from just one fish it is possible to generate at least 40 primary gill cultures (Fig. 34.2). Liver spheroids have been shown to retain several tissue-specific features, including cellular morphology, glucose and albumin synthesis, and several cytochrome enzymes.32,37,38 Liver spheroids have also been shown to metabolize pharmaceuticals more efficiently than liver homogenate, known as S9 fractions.37 Thus, coculture of primary gill on transwells and primary liver spheroids on the sublocated well might represent an effective in vitro model to determine the uptake,
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34.2 Alternative methods in regulatory ecotoxicology
bioaccumulation, and biotransformation of xenobiotics. On the other hand, the preparation of primary cultures brings also some disadvantages. First of all there is a need for live specimens for the isolation of primary cells. Second the preparation of primary cultures is time consuming, laborious, and often produces results that are variable.39 Therefore the use of continuous cell lines, which do not show the limitations described earlier, might be preferable. 34.2.2.2 Continuous cell lines Primary cultures are isolated from tissues and organs taken directly from living organisms. Most cells derived from a primary culture will eventually become senescent and die. However, if a primary culture starts to proliferate in vitro and can be divided and propagated into new culture flasks, it becomes a cell line. If a cell line can be propagated for a limited time, it is finite, or if it can be propagated indefinitely it becomes an immortal or continuous cell line (Fig. 34.2). According to Bols et al.,39 mammalian cell lines are finite or continuous, whereas most fish cell lines appear to be continuous. Cell lines have been isolated from several fish tissues including gill, liver, kidney, intestine, gonads, and connective tissue. One cell line, the RTgill-W1 isolated by the Bols laboratory in 1994 from the rainbow trout (O. mykiss) gill,40 represents a promising alternative to the fish acute toxicity test (OECD test guideline 203). As mentioned earlier impairment of the gill tissue after acute toxicant exposure is linked with fish death. Thus RTgill-W1 represents an ideal alternative model because it can link cellular gill toxicity with fish mortality. It has been shown that impairment of cellular endpoints, such as cell metabolic activity, cell membrane integrity and cell lysosomal integrity, in cells exposed to organic and inorganic chemicals correlates well to mortality in fish exposed to the same chemicals.10,41
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Furthermore this method has shown high repeatability (low intralaboratory variability) and reproducibility (low interlaboratory variability) in predicting the toxicity of organic chemicals.19 In addition to the RTgill-W1 cell line, which is particularly relevant to study and test the toxicity of chemicals dissolved in water, other cell lines should be mentioned. The RTgutGC cell line isolated from the rainbow trout (O. mykiss) intestine is relevant to study chemical uptake and toxicity via the dietary route.42 The RTgutGC cell line was shown to conserve several features of the polarized intestinal epithelium in vivo including apical expression of tight junction proteins and basolateral expression of the Na/K-ATPase.43 It was also shown that RTgutGC cells express alkaline phosphatase and cytochrome P450 enzymes, which are key enzymes for intestinal immune and metabolic function.42,44 RTgutGC cells have been used for ecotoxicology, immunology, fish nutrition, and physiology. Several cell lines have been isolated from the fish hepatic tissue because of its key role in chemical metabolism and bioaccumulation.45 47 The RTL-W1 cell line, derived from the rainbow trout (O. mykiss) liver, has shown cytochrome p450 activity and has been used extensively for the study of biotransformation, bioconcentration, and hepatic toxicity of chemicals.44,46 Moreover RTL-W1 spheroids show higher CYP1A activity compared to cells cultured in 2D.48 The use of fish cell lines has been proposed as an alternative to acute fish toxicology testing.17,49 Cell lines from vertebrates are ideal replacement alternatives to whole animal testing in toxicology as once the cell lines have been established there is no further need for live animals. Cell lines can be frozen indefinitely and thawed when needed. Several cell lines are readily available through commercial purchase. They are easier and less expensive to maintain than live specimens, and they can be used for high-throughput approaches.
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However, cell lines present some limitations that should be considered and addressed. Cell lines may be less sensitive than whole animals to the toxicity of certain chemicals. This could be the case when the mode of toxic action of a chemical in an animal involves the inhibition of a specific receptor or pathway not present in the cell line. For example, it was shown that neurotoxicants do not induce toxicity in RTgill-W1 cells.10,41 In other cases the reduced sensitivity to chemicals can be due to the exposure conditions. Cell lines are normally cultured in complex media containing salts, vitamins, amino acids, and proteins that can complex with chemicals and thus reduce bioavailability. Although the exposure media can be simplified to reduce such interactions, cell lines cannot tolerate exposure in a media of low osmolarity such as fresh water. The medium is thus adjusted by adding salts to increase the media osmolarity, but salts can also reduce the chemical bioavailability, especially if the chemical evaluated is a metal. In other cases nonpolar or volatile chemicals can partition out of the in vitro exposure system (i.e., plastic multiwell plates) thus affecting their bioavailability. In summary, cell lines are a promising tool for regulatory toxicology but require thorough characterization of the cell line physiology and biochemistry. Moreover the exposure conditions should be carefully considered when assessing the chemical bioavailability and toxicity. 34.2.2.3 New frontiers for in vitro models in ecotoxicology The development of novel culturing techniques designed to maintain a more physiological cellular microenvironment and to maintain the chemical bioavailable in the exposure system will be instrumental to improve in vitro models in ecotoxicology. Therefore the new frontier for the development of improved in vitro models in toxicology is the engineering of new biocompatible
materials and/or exposure systems. For example, the lung-on-a-chip uses microfluidics and elastic permeable membranes to recreate the complex alveolar microenvironment resulting in a cellular response that is remarkably close to the one of the lung tissue in vivo.50 The use of ultrathin aluminum oxide permeable membranes showed improved permeability and transparency in comparison to the commercially available PET membranes, allowing a more physiological culturing environment and improved microscopy capabilities in RTgutGC.51 The study of volatile and hydrophobic chemicals has proven problematic due to the tendency of these chemicals to partition out of the exposure system, to stick to plastic material and generally for their low solubility in aqueous medium. All these aspects complicate the exposure conditions. While these are problems that can also affect the exposure in vivo, utilizing novel exposure chamber technologies, such as headspace-free setups,52 and cellular barrier transfer systems, such as TransFEr,53 has proven beneficial to test the effect of volatile and hydrophobic organic chemicals in vitro cell assays.
34.3 Conclusion Environmental agencies around the world agree on the need to reduce the use of animals for regulatory ecotoxicology testing. There is already a large body of data showing that FETAX, FETs and cell line cytotoxicity assays can be used to determine toxicity of chemicals dissolved or dispersed in water. These assays serve as a valid alternative to whole animal testing. Moreover such in vitro alternatives allow a better understanding of chemicals mode of toxic action. However, the use of alternatives is still very limited. More can be done by the academic community to develop more reliable, cost effective, and informative in vitro assays. But ultimately, reduction or replacement of animal use
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References
in regulatory toxicology should be led by government, industry, and potentially by public demand.
Acknowledgments J.S. was funded by a grant from the Alternatives Research & Development Foundation (ARDF No. G1000158).
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14. OECD. Validation report (phase 2) for the zebrafish embryo toxicity test; 2012. 15. OECD. Guideline for Testing of Chemicals, 203. Fish, Acute Toxicity Test; 1992. 16. Busquet F, et al. OECD validation study to assess intraand inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing. Regul Toxicol Pharmacol 2014;69:496 511. 17. Schirmer K. Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicology 2006;224:163 83. 18. Dayeh VR, Bols NC, Tanneberger K, Schirmer K, Lee LEJ. The use of fish-derived cell lines for investigation of environmental contaminants: an update following OECD’s fish toxicity testing framework no. 171. Curr Protoc Toxicol 2013;1. 19. Fischer M, et al. Repeatability and reproducibility of the RTgill-W1 cell line assay for predicting fish acute toxicity. Toxicol Sci 2019;1 12. Available from: https:// doi.org/10.1093/toxsci/kfz057. 20. OECD. Fish toxicity testing framework. Series on testing and assessment. Organ Econ Co-operation Dev 2012;1 174. 21. Patwardhan V, Ghaskadbi S. Invertebrate alternatives for toxicity testing: hydra stakes its claim. Proc Anim Altern Teaching Toxic Test Med 2013;1:69 76. 22. Guilhermino L, Diamantino T, Carolina Silva M, Soares AMVM. Acute toxicity test with Daphnia magna: an alternative to mammals in the prescreening of chemical toxicity? Ecotoxicol Environ Saf 2000;46:357 62. 23. Ohta T, Tokishita SI, Shiga Y, Hanazato T, Yamagata H. An assay system for detecting environmental toxicants with cultured cladoceran eggs in vitro: malformations induced by ethylenethiourea. Environ Res 1998;77:43 8. 24. Halder M, et al. Regulatory aspects on the use of fish embryos in environmental toxicology. Integr Environ Assess Manag 2010;6:484 91. 25. US-EPA. Method 1001.0: Fathead minnow, pimephales promelas, larval survival and teratogenicity test; chronic toxicity; 2002. 26. Fort DJ, Stover EL, Farmer DR, Lemen JK. Assessing the predictive validity of frog embryo teratogenesis assay - Xenopus (FETAX). Teratog Carcinog Mutagen 2000;20:87 98. 27. Bantle J, Sabourin T, Standard guide for conducting the frog embryo teratogenesis assay-Xenopus (FETAX), Am Soc Test Mater E1439. 1991;98:1 20. 28. Bantle JA, Fort DJ, James BL. Identification of developmental toxicants using the frog embryo teratogenesis assayxenopus (FETAX). Hydrobiologia 1989;188 189:577 85. 29. Lopez-Luna J, Al-Jubouri Q, Al-Nuaimy W, Sneddon LU. Reduction in activity by noxious chemical stimulation is ameliorated by immersion in analgesic drugs in zebrafish. J Exp Biol 2017;220:1451 8.
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30. Burke J, Handy RD. Sodium-sensitive and -insensitive copper accumulation by isolated intestinal cells of rainbow trout Oncorhynchus mykiss. J Exp Biol 2005;208: 391 407. 31. Bury NR, Schnell S, Hogstrand C. Gill cell culture systems as models for aquatic environmental monitoring. J Exp Biol 2014;217:639 50. 32. Baron MG, Purcell WM, Jackson SK, Owen SF, Jha AN. Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes. Ecotoxicology 2012;21:2419 29. 33. Schnell S, et al. Procedures for the reconstruction, primary culture and experimental use of rainbow trout gill epithelia. Nat Protoc 2016;11:490 8. 34. Evans DH, Piermarini PMPM, Choe KPKP. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 2005;85:97 177. 35. Minghetti M, Schnell S, Chadwick MA, Hogstrand C, Bury NR. A primary fish gill cell system (FIGCS) for environmental monitoring of river waters. Aquat Toxicol 2014;154:184 92. 36. Stott LC, Schnell S, Hogstrand C, Owen SF, Bury NR. A primary fish gill cell culture model to assess pharmaceutical uptake and efflux: evidence for passive and facilitated transport. Aquat Toxicol 2014;159:127 37. 37. Baron MG, et al. Pharmaceutical metabolism in fish: using a 3-D hepatic in vitro model to assess clearance. PLoS One 2017;12:1 13. 38. Uchea C, Owen SF, Chipman JK. Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures. Toxicol Res (Camb) 2015;4:494 507. 39. Bols N, Dayeh VR, Lee LEJ, Schirmer K. Use of fish cell lines in the toxicology and ecotoxicology of fish. Biochem Mol Biol Fishes 2005;6:43 85. 40. Bols NC, Barlian A, Chirino-Trejo M, Caldwell SJ. Development of a cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills. J Fish Dis 1994;17:601 11. 41. Scott J. Optimization of the fish gill cell line, RTgill-W1 for use in acute whole effluent toxicity testing. Oklahoma State University; 2018. 42. Kawano A, et al. Development of a rainbow trout intestinal epithelial cell line and its response to lipopolysaccharide. Aquac Nutr 2011;17:e241 52.
43. Minghetti M, Drieschner C, Bramaz N, Schug H, Schirmer K. A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC. Cell Biol Toxicol 2017;33: 539 55. 44. Stadnicka-Michalak J, Weiss FT, Fischer M, Tanneberger K, Schirmer K. Biotransformation of benzo[a]pyrene by three rainbow trout (Onchorhynchus mykiss) cell lines and extrapolation to derive a fish bioconcentration factor. Environ Sci Technol 2018;52: 3091 100. 45. Scholz S, Braunbeck T, Segner H. Viability and differential function of rainbow trout liver cells in primary culture: coculture with two permanent fish cells. Vitr Cell Dev Biol Anim 1998;34:762 71. 46. Lee LE, et al. Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity. Cell Biol Toxicol 1993;9:279 94. 47. Franco ME, Sutherland GE, Lavado R. Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: biomarkers of CYP450 activity and oxidative stress. Comp Biochem Physiol Pt C Toxicol Pharmacol 2018;206 207: 32 40. 48. Lammel T, Tsoukatou G, Jellinek J, Sturve J. Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1. Ecotoxicol Environ Saf 2019;167:250 8. 49. Segner H. Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish. Altern Lab Anim 2004;32:375 82. 50. Huh D, et al. A human disease model of drug toxicityinduced pulmonary edema in a lung-on-a-chip microdevice. Sci Transl Med 2012;4:159ra147. 51. Drieschner C, Minghetti M, Wu S, Renaud P, Schirmer K. Ultrathin alumina membranes as scaffold for epithelial cell culture from the intestine of rainbow trout. ACS Appl Mater Interf 2017;9:9496 505. 52. Stalter D, Dutt M, Escher BI. Headspace-free setup of in vitro bioassays for the evaluation of volatile disinfection by-products. Chem Res Toxicol 2013;26:1605 14. 53. Schug H, et al. TransFEr: a new device to measure the transfer of volatile and hydrophobic organic chemicals across an in vitro intestinal fish cell barrier. Anal Methods 2018;10:4394 403.
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34 Toxicity testing: in vitro models in ecotoxicology Justin Scott and Matteo Minghetti Department of Integrative Biology, Oklahoma State University, Stillwater, OK, United States
34.1 Overview of the use of animals in toxicology 34.1.1 Use of animals in scientific research: historic perspective Animals were used by early Greek philosophers and physicians such as Aristotle (384 322 BCE) and Galen (CE 129 210) to advance our understanding of anatomy, physiology, pathology, and toxicology. Since then advancements in these disciplines have relied heavily on animal experimentation. However, while the use of animals in scientific research has undoubtedly contributed to the advancement of biomedical research, it has also raised ethical concerns. The debate on the ethical grounds for animal experimentation is depicted in an iconic painting from Joseph Wright “An Experiment on a Bird in the Air Pump” in 1768.1 The painting depicts a natural philosopher, recreating one of Robert Boyle’s air pump experiments, in which a bird is deprived of air before a group of observers. The group exhibits a variety of reactions from shock to curiosity. What is most relevant about
An Introduction to Interdisciplinary Toxicology DOI: https://doi.org/10.1016/B978-0-12-813602-7.00034-X
the painting is its illustration of the variety of reactions that animal experimentation can induce in the general public. In modern times academic debate, pressure from the general public, and an increase in animal rights’ movements have resulted in government legislation that protects animal welfare. For instance, in the United States, the Institutional Animal Care and Use Committees and in the United Kingdom the Animals (Scientific Procedures) Act were formally instituted in 1986 to review and approve all government funded animal research. Around one hundred million vertebrate animals are used worldwide annually for experimental and regulatory purposes.2 Over 50% of these animals are mice, while fish account for around 10%; a figure that has been increasing over the past 15 years.3 Russell and Burch’s The Principles of Humane Experimental Technique 1959 was pivotal in emphasizing a need for alternative approaches to animal testing and introducing the concept of the “3Rs” (reduction, replacement, and refinement). Over the past three decades, there has been increased interest among the academic, regulatory, and
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private sector communities toward in vitro alternatives to vertebrate animals used for ecotoxicity testing. In vitro alternatives consist of isolated vertebrate cells, biochemical tests, and computer models. A focus exists to move away from, and eliminate altogether, the use of live vertebrate testing specimens due to increasing ethical concerns and the necessity for efficient and less expensive high-throughput testing alternatives. For instance in 2013 the European Commission banned cosmetics, personal care products, and ingredients tested on animals. This process took 20 years of debate between regulators, academia, and industry and was made possible by the development of efficient in vitro alternatives and by sustained public pressure.4,5 Currently most regulatory methods in ecotoxicology still use vertebrate animals (mainly fish) and lethality as a measured endpoint to assess the toxicity of chemicals. Classical acute toxicity testing requires the calculation of Lethal Dose 50% (LD50). The value of LD50 for a substance is the dose required to kill half of a tested population after a specified test duration. Such methods are popular for the ease of execution and interpretation, but do not necessarily generate useful information to predict the substance’s mode of toxic action. Therefore the development of alternatives to whole animal testing, which allow the study of the mechanisms of toxicity in a more ethical and cost-effective manner, is relevant and timely.
34.1.2 Alternatives to animal testing in ecotoxicology Ecotoxicology focuses on preventing, monitoring, and alleviating harmful effects of anthropogenic activities on the environment. Due to the increased number and volume of chemicals developed for societal and industrial progress, pressure on the environment is increasing. A major concern is the negative
impact on the aquatic environment. Contributing sectors are: agriculture, pharmaceutical and chemical production, petrochemical refinement, mining operations, animal farming, power and utility operations, as well as man-made ecological disasters. Environmental protection agencies in most of the western world have developed legislation to routinely monitor and assess the state of the aquatic environment (e.g. Clean Water Act— USA; Water Frame Directive—EU). In addition, specific legislation has been developed to regulate the registration of new chemicals and evaluate potential environmental impacts.6,7 While there are differences in the standards and guidelines across borders, the shared goal to reduce and/or eliminate anthropogenic effects on the environment has also now been extended to reducing the use of vertebrae animals in toxicity testing.6,8 The US Environmental Protection Agency (EPA) has initiated the Toxicity Forecaster (ToxCast) and Aggregated Computational Toxicology Resource, which uses in vitro highthroughput screening approaches and computational toxicology approaches to rank and prioritize chemical toxicity.9 Other organizations, including the Alternatives Research and Development Foundation (ARDF), Interagency Coordinating Committee on the Validation of Alternative Methods, and European Centre for Validation of Alternative Methods, are also pursuing advancements in toxicity testing to identify and validate in vitro alternatives to reduce the number of live organisms for in vivo testing. However, most toxicity testing for the determination of potentially harmful chemicals still relies on the need for live vertebrates. For instance, in the United States alone, three million fish are used every year for Whole Effluent Toxicity (WET) testing.10 Several in vitro alternatives have been developed to determine the ecotoxicity of chemicals; however, the validation of alternative methods remains critical to allow the use of such
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34.2 Alternative methods in regulatory ecotoxicology
in vitro alternatives in regulatory toxicology. Specifically, there is a need to provide regulatory agencies, whose focus is to enforce methods aimed at preventing and monitoring harmful anthropogenic activities, with reliable and reproducible results. The aim of this chapter is to describe the current state of in vitro alternatives highlighting their strengths and weaknesses and suggest future directions to validate the most promising alternative methods in regulatory ecotoxicity testing.
34.2 Alternative methods in regulatory ecotoxicology In vitro alternative methods approved for regulatory purposes include the fish embryo test (FET), approved by the Organisation for Economic Co-operation and Development (OECD) and by the International Organization for Standardization (ISO) (OECD 236; ISO 1508811,12) and the fish gill cell line (RTgill-W1) approved by ISO (ISO 21115-201913). The OECD approval process of the FET took nearly a decade and involved two phases. Phase 1 aimed to evaluate the transferability and the intra- and interlaboratory reproducibility of the FET after exposure to seven chemicals. Phase 2 built on this by further testing reproducibility with the addition of 13 chemicals and covering specific areas of use (i.e., chemicals, pharmaceuticals, pesticides, and biocides).14 This process resulted in the approval of the use of the FET to replace the in vivo acute toxicity test (OECD Guideline 20315) for regulatory testing.16 The ISO standard 15088 has also been developed as a substitute for the acute fish toxicity test (ISO 7346-1 and 7346-2) when applied to waste water. Additionally the use of cytotoxicity assays using fish cell lines (see Section 34.2.2.2) has been proposed as a viable alternative to the fish acute toxicity test.17 19 The recent determination of the rainbow trout
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(Oncorhynchus mykiss) gill cell line, RTgill-W1, as an in vitro alternative to the fish acute toxicity test resulted in the approval of the method ISO 21115-201913. Moreover, the demonstration of inter- and intra-laboratory reproducibility of the RTgill-W1 cell assay19 places this method a step closer to OECD approval. Mathematical models and the use of invertebrates have also been used as valid alternatives to vertebrates in regulatory toxicology. Quantitative structure activity relationship models (QSAR) are mathematical models used by European and North American legislations to predict both bioaccumulation and short- and long-term toxicity in fish. QSAR are nontesting models used to predict toxicity of chemicals based on their physical and chemical properties and on the principle that similar substances have similar biological activity. QSAR predictions for acute toxicity and bioconcentration in fish are commonly used and well accepted for regulatory purposes (OECD 17120). Additionally among the alternative methods under investigation, the use of invertebrates, such as the Hydra21 and the Daphnia,22,23 can effectively detect the toxicity of chemicals dissolved in water. However, the simplicity of these organisms, while being a methodological strength, is also a limitation, since their physiology is much less complex than that of vertebrate animals. Nonetheless, they represent good alternative models for preliminary toxicity screening, which can substantially reduce the use of vertebrate animals. Tiered testing approaches for chemical registration, risk assessment, compliance with exposure levels, and fate of contaminants all use aquatic toxicity testing components with both vertebrate and invertebrate in vivo models. The following alternative model systems have been chosen for their use and significance in regulatory ecotoxicology and for their potential development into an approved alternative approach to whole vertebrate animal testing.
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FIGURE 34.1 Panel A illustrates the zebrafish (Danio rerio), the fathead minnow (Pimephales promelas), and the African clawed frog (Xenopus laevis) developmental stages relevant for embryo toxicity and teratogenicity assays. According to the Organisation for Economic Co-operation and Development, fish and amphibian embryos/larvae are considered nonprotected until the animal is capable of independent feeding, occurring approximately at 120, .176, and 96 h postfertilization for the zebrafish, fathead minnow, and Xenopus, respectively. Panel B illustrates the measured toxicological endpoints for the zebrafish fish embryo test (i.e., coagulation of the embryos; lack of somite formation; nondetachment of the tail; lack of heartbeat), which are measured at 24 h intervals until test termination.
34.2.1 Fish and amphibian embryos The FET and the Frog Embryo Teratogenesis Assay-Xenopaus (FETAX) can be defined as alternatives to animal tests based on a convention that establishes that embryos are not protected animals up to a certain stage of development and can serve as a viable in vitro alternative. The OECD’s concept of a protected animal is defined as any living vertebrate, other than man, at the point in which it becomes capable of independent feeding. Therefore organisms at embryonic and eleuthero-embryonic stages, where the organism is still using the yolk sac as
energy source, are not protected animals. The US Office of Laboratory Animal Welfare sets a more stringent definition for fish and amphibians and considers hatching as the point at which organisms are protected.24 Fig. 34.1 shows and compares fish and amphibian embryo life cycles with their respective method for ecotoxicology. The FET has gained promising strides both in Europe with the use of zebrafish FET as an alternative to acute fish testing, and in the United States with the EPA’s use of fathead minnows’ larval survival and teratogenicity tests and chronic toxicity.25 The FET is also utilized for several regulatory
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FIGURE 34.2 Preparation of gill cell cultures from rainbow trout (Oncorhynchus mykiss). (A C) Gill arches are excised, gill filaments are separated and washed in phosphate buffer. (D) Gill cells are washed repeatedly with antibiotic and antifungal solutions and dissociated from each other using a cell strainer and enzymatic digestion (i.e., trypsin digestion). (E) Cell viability and total numbers are evaluated using the trypan blue exclusion assay with a hemocytometer or automatic cell counter. (F) The primary gill cell culture system is generated by seeding two primary gill cells cultures (steps A E) on a polyethylene terephthalate porous membranes in two consequent days. (G) Double-seeded primary gill cultures form a tight epithelium when cultured in symmetrical conditions for 5 7 days and when a trans epithelial electrical resistance of above 5000 Ω cm2 is reached cells can be maintained in asymmetric conditions and tolerate direct exposure to fresh water. (H) A gill cell line was isolated using a similar protocol (steps A E). Primary gill cells were cultured as monolayers on culture flasks and passaged several times until the RTgill-W1 cell line was isolated. (I) RTgill-W1 cells can be cryopreserved and thawed when needed for cytotoxicity assays and other applications.
biomonitoring protocols including the International Maritime Dangerous Goods Code, Federal Insecticide Fungicide and Rodenticide Act, and WET testing methods to detect chronic effects. The FET allows toxicological observations to be made on the organism at the earliest stages of development. Embryos are gathered postfertilization from tanks holding spawning adult breeders and then exposed to aquatic samples to help measure and determine toxicological impacts. Viability of each embryo is visually observed with a microscope at
24 hours intervals over the test duration (acute at 48 and 96 hours postfertilization) to measure the toxicity markers which include: (1) coagulation of the embryos; (2) lack of somite development; (3) nondetachment of the tail, and; (4) lack of heartbeat which is used as endpoint of mortality and also to generate Lethal Concentrations 50% (LC50) values. The FETAX is a method similar to FETs and developed using the African clawed frog (Xenopus laevis) from the amphibian metamorphosis test. The FETAX assay allows monitoring of lethal and sublethal endpoints through a more
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cost effective and expedited acute assay using in vitro approaches.26 Using fertilized frog embryos, three endpoints are evaluated (mortality, malformations, and embryonic growth). Similarly to the FET assay, observations are recorded at 48, 72, and 96 hours, and lack of heartbeat is used to generate LC50 values.27 FETAX studies have been used to assess and predict both ecological and human health hazard evaluations.28 From an ethical point of view, the FET and FETAX do not truly take on the concept of live animal toxicity testing replacement, but rather significantly reduce the number of animals used and refine an approach to measure additional toxicological endpoints. In addition, the idea of fish embryo and larvae welfare has been considered when used in toxicity testing. For instance it has been found that 120 hours postfertilization fish larvae responded to noxious stimuli and that the response was relieved with pain reducing drugs.29 Moreover although FET and FETAX have shown to be effective predictive methods of ecotoxicity of chemicals, they require husbandry practices of breeding adult vertebrate test specimens which have ethical and cost implications.
34.2.2 Use of isolated fish cells 34.2.2.1 Primary cultures Primary cell cultures are prepared by excising a selected tissue and by maintaining the isolated tissue cells in vitro. Primary cells retain most of the tissue cellular physiology but remain viable for a relatively short time, from a few hours to a few weeks. Cell viability should be monitored to determine the extent of time primary cells can be used for experiments. For instance, intestinal cells isolated from rainbow trout (O. mykiss) were shown to remain viable for 4 hours using the trypan blue exclusion assay.30 On the other hand, primary gill cells cultured on permeable membrane
remained viable for at least 2 weeks and primary hepatocytes prepared to form spheroid structures remained viable for up to 4 weeks.31,32 Culturing techniques designed to maintain the in vivo cellular architecture and microenvironment, such as culturing cells on porous membranes or allowing the formation of 3D structures (e.g., spheroids), have also been shown to allow better retention of the in vivo tissue-specific features in comparison to cell monolayers (i.e., 2D cultures). Primary gill cells cultured on polyethylene terephthalate (PET) porous membranes retain several of the features of the gill epithelium in vivo, including a high trans epithelial electrical resistance (over 20 kΩ cm2), expression of apical tight junction protein and presence of mitochondria rich cells which are key feature of this tissue.31,33 In fish the gill is extremely sensitive to environmental insults and serves as a multifunctional organ. Considering its large surface area, it is the major site of toxicant exposure and uptake.34 The cost associated with gill failure is inevitably the death of the organism. Thus the gill represents an ideal model to be used as an alternative to acute fish toxicity testing. Primary gill cells have been used for fish physiology and toxicology studies including biomonitoring of pollutants in river waters.31,35,36 Moreover with the 3Rs in mind, the use of primary gill cells reduces the number of fish used for acute toxicity tests, as from just one fish it is possible to generate at least 40 primary gill cultures (Fig. 34.2). Liver spheroids have been shown to retain several tissue-specific features, including cellular morphology, glucose and albumin synthesis, and several cytochrome enzymes.32,37,38 Liver spheroids have also been shown to metabolize pharmaceuticals more efficiently than liver homogenate, known as S9 fractions.37 Thus, coculture of primary gill on transwells and primary liver spheroids on the sublocated well might represent an effective in vitro model to determine the uptake,
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34.2 Alternative methods in regulatory ecotoxicology
bioaccumulation, and biotransformation of xenobiotics. On the other hand, the preparation of primary cultures brings also some disadvantages. First of all there is a need for live specimens for the isolation of primary cells. Second the preparation of primary cultures is time consuming, laborious, and often produces results that are variable.39 Therefore the use of continuous cell lines, which do not show the limitations described earlier, might be preferable. 34.2.2.2 Continuous cell lines Primary cultures are isolated from tissues and organs taken directly from living organisms. Most cells derived from a primary culture will eventually become senescent and die. However, if a primary culture starts to proliferate in vitro and can be divided and propagated into new culture flasks, it becomes a cell line. If a cell line can be propagated for a limited time, it is finite, or if it can be propagated indefinitely it becomes an immortal or continuous cell line (Fig. 34.2). According to Bols et al.,39 mammalian cell lines are finite or continuous, whereas most fish cell lines appear to be continuous. Cell lines have been isolated from several fish tissues including gill, liver, kidney, intestine, gonads, and connective tissue. One cell line, the RTgill-W1 isolated by the Bols laboratory in 1994 from the rainbow trout (O. mykiss) gill,40 represents a promising alternative to the fish acute toxicity test (OECD test guideline 203). As mentioned earlier impairment of the gill tissue after acute toxicant exposure is linked with fish death. Thus RTgill-W1 represents an ideal alternative model because it can link cellular gill toxicity with fish mortality. It has been shown that impairment of cellular endpoints, such as cell metabolic activity, cell membrane integrity and cell lysosomal integrity, in cells exposed to organic and inorganic chemicals correlates well to mortality in fish exposed to the same chemicals.10,41
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Furthermore this method has shown high repeatability (low intralaboratory variability) and reproducibility (low interlaboratory variability) in predicting the toxicity of organic chemicals.19 In addition to the RTgill-W1 cell line, which is particularly relevant to study and test the toxicity of chemicals dissolved in water, other cell lines should be mentioned. The RTgutGC cell line isolated from the rainbow trout (O. mykiss) intestine is relevant to study chemical uptake and toxicity via the dietary route.42 The RTgutGC cell line was shown to conserve several features of the polarized intestinal epithelium in vivo including apical expression of tight junction proteins and basolateral expression of the Na/K-ATPase.43 It was also shown that RTgutGC cells express alkaline phosphatase and cytochrome P450 enzymes, which are key enzymes for intestinal immune and metabolic function.42,44 RTgutGC cells have been used for ecotoxicology, immunology, fish nutrition, and physiology. Several cell lines have been isolated from the fish hepatic tissue because of its key role in chemical metabolism and bioaccumulation.45 47 The RTL-W1 cell line, derived from the rainbow trout (O. mykiss) liver, has shown cytochrome p450 activity and has been used extensively for the study of biotransformation, bioconcentration, and hepatic toxicity of chemicals.44,46 Moreover RTL-W1 spheroids show higher CYP1A activity compared to cells cultured in 2D.48 The use of fish cell lines has been proposed as an alternative to acute fish toxicology testing.17,49 Cell lines from vertebrates are ideal replacement alternatives to whole animal testing in toxicology as once the cell lines have been established there is no further need for live animals. Cell lines can be frozen indefinitely and thawed when needed. Several cell lines are readily available through commercial purchase. They are easier and less expensive to maintain than live specimens, and they can be used for high-throughput approaches.
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However, cell lines present some limitations that should be considered and addressed. Cell lines may be less sensitive than whole animals to the toxicity of certain chemicals. This could be the case when the mode of toxic action of a chemical in an animal involves the inhibition of a specific receptor or pathway not present in the cell line. For example, it was shown that neurotoxicants do not induce toxicity in RTgill-W1 cells.10,41 In other cases the reduced sensitivity to chemicals can be due to the exposure conditions. Cell lines are normally cultured in complex media containing salts, vitamins, amino acids, and proteins that can complex with chemicals and thus reduce bioavailability. Although the exposure media can be simplified to reduce such interactions, cell lines cannot tolerate exposure in a media of low osmolarity such as fresh water. The medium is thus adjusted by adding salts to increase the media osmolarity, but salts can also reduce the chemical bioavailability, especially if the chemical evaluated is a metal. In other cases nonpolar or volatile chemicals can partition out of the in vitro exposure system (i.e., plastic multiwell plates) thus affecting their bioavailability. In summary, cell lines are a promising tool for regulatory toxicology but require thorough characterization of the cell line physiology and biochemistry. Moreover the exposure conditions should be carefully considered when assessing the chemical bioavailability and toxicity. 34.2.2.3 New frontiers for in vitro models in ecotoxicology The development of novel culturing techniques designed to maintain a more physiological cellular microenvironment and to maintain the chemical bioavailable in the exposure system will be instrumental to improve in vitro models in ecotoxicology. Therefore the new frontier for the development of improved in vitro models in toxicology is the engineering of new biocompatible
materials and/or exposure systems. For example, the lung-on-a-chip uses microfluidics and elastic permeable membranes to recreate the complex alveolar microenvironment resulting in a cellular response that is remarkably close to the one of the lung tissue in vivo.50 The use of ultrathin aluminum oxide permeable membranes showed improved permeability and transparency in comparison to the commercially available PET membranes, allowing a more physiological culturing environment and improved microscopy capabilities in RTgutGC.51 The study of volatile and hydrophobic chemicals has proven problematic due to the tendency of these chemicals to partition out of the exposure system, to stick to plastic material and generally for their low solubility in aqueous medium. All these aspects complicate the exposure conditions. While these are problems that can also affect the exposure in vivo, utilizing novel exposure chamber technologies, such as headspace-free setups,52 and cellular barrier transfer systems, such as TransFEr,53 has proven beneficial to test the effect of volatile and hydrophobic organic chemicals in vitro cell assays.
34.3 Conclusion Environmental agencies around the world agree on the need to reduce the use of animals for regulatory ecotoxicology testing. There is already a large body of data showing that FETAX, FETs and cell line cytotoxicity assays can be used to determine toxicity of chemicals dissolved or dispersed in water. These assays serve as a valid alternative to whole animal testing. Moreover such in vitro alternatives allow a better understanding of chemicals mode of toxic action. However, the use of alternatives is still very limited. More can be done by the academic community to develop more reliable, cost effective, and informative in vitro assays. But ultimately, reduction or replacement of animal use
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References
in regulatory toxicology should be led by government, industry, and potentially by public demand.
Acknowledgments J.S. was funded by a grant from the Alternatives Research & Development Foundation (ARDF No. G1000158).
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43. Minghetti M, Drieschner C, Bramaz N, Schug H, Schirmer K. A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC. Cell Biol Toxicol 2017;33: 539 55. 44. Stadnicka-Michalak J, Weiss FT, Fischer M, Tanneberger K, Schirmer K. Biotransformation of benzo[a]pyrene by three rainbow trout (Onchorhynchus mykiss) cell lines and extrapolation to derive a fish bioconcentration factor. Environ Sci Technol 2018;52: 3091 100. 45. Scholz S, Braunbeck T, Segner H. Viability and differential function of rainbow trout liver cells in primary culture: coculture with two permanent fish cells. Vitr Cell Dev Biol Anim 1998;34:762 71. 46. Lee LE, et al. Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity. Cell Biol Toxicol 1993;9:279 94. 47. Franco ME, Sutherland GE, Lavado R. Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: biomarkers of CYP450 activity and oxidative stress. Comp Biochem Physiol Pt C Toxicol Pharmacol 2018;206 207: 32 40. 48. Lammel T, Tsoukatou G, Jellinek J, Sturve J. Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1. Ecotoxicol Environ Saf 2019;167:250 8. 49. Segner H. Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish. Altern Lab Anim 2004;32:375 82. 50. Huh D, et al. A human disease model of drug toxicityinduced pulmonary edema in a lung-on-a-chip microdevice. Sci Transl Med 2012;4:159ra147. 51. Drieschner C, Minghetti M, Wu S, Renaud P, Schirmer K. Ultrathin alumina membranes as scaffold for epithelial cell culture from the intestine of rainbow trout. ACS Appl Mater Interf 2017;9:9496 505. 52. Stalter D, Dutt M, Escher BI. Headspace-free setup of in vitro bioassays for the evaluation of volatile disinfection by-products. Chem Res Toxicol 2013;26:1605 14. 53. Schug H, et al. TransFEr: a new device to measure the transfer of volatile and hydrophobic organic chemicals across an in vitro intestinal fish cell barrier. Anal Methods 2018;10:4394 403.
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