Chemical Lab Testing is Boosted by Unique Fish Species

November 1, 2019

Written By

Sue Cielinski

As you consider your chemical lab testing needs, investigating new species can unleash a better fit for assessment of bioaccumulation prediction. KJ Scientific is leading progress on in vitro metabolism testing by making a better prediction of environmental health across species.

The metabolism and removal of a foreign chemical is directly related to whether the chemical is toxic to an organism. Toxicokinetics, the study of essentially “how a chemical gets into the body and what happens to it when it’s in the body” looks at the relationship of chemical exposure of animals and how they may correspond to exposure in humans. One of the key processes that happens to chemicals in the body is biotransformation or metabolism, transformation of the chemical into new chemicals. Some chemicals are inactivated by metabolism while others bioaccumulate or become more of a problem.

In practice animals are used for chemical lab testing, for example, fish, rats and mice, are exposed by giving doses of test chemicals in various ways such as submersion, oral, and intravenous. The animals are then put down and examined for the accumulation of test chemicals in their organs. In fish as in mammals, the liver is the principal organ of the body that processes chemicals by undergoing biotransformation and helps removal from the body. The differences between species and the amount and type of liver enzymes for metabolism make it challenging to extrapolate modeling for environmental chemical risk assessment. KJ Scientific’s innovative work with liver S9 fraction and hepatocytes of uncharted fish species is making headway on assessment of metabolism of chemicals for human benefit.

Regulatory Guidelines for Chemical Lab Testing

Regulatory agencies in developed countries around the world require chemical lab testing for bioaccumulation of hydrophobic (repel and separate out of water) organic chemicals including petrochemical products, pesticides, cosmetic ingredients in consumer goods and pharmaceuticals. Chemical lab testing is required for the production and use of chemicals and potential impacts to environmental and human health for example through the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) in Europe. REACH addresses whether a company uses, produces, are in Europe or deal with Europe to address persistency, bioaccumulation and toxicity (PBT) of their products. There are thousands of chemicals that need to be tested, and new chemicals are being developed all the time.

The U.S. Environmental Protection Agency (EPA) has guidelines for chemical lab testing for toxicity of pesticides and chemicals for potential to be taken in or bioconcentrate in tissues of fish. These tests are used to help measure risks to fish and to other organisms as well as mammalian species. EPA favors test fish species easily available and maintained in the laboratory and are of recreational, commercial, and ecological importance haven been successfully used in the past. Some well-known and popular freshwater test fish species are rainbow trout, bluegill, sunfish, zebra-fish, fathead minnow, and common carp. Some estuarine and marine species used are sheepshead minnow, silverside, three-spined stickleback, and sea bass. The fish are exposed to the chemical, mortality and harmful effects are reported and any change in behavior is observed. The fish tissue is tested for the concentration of the test chemical and the natural lipid content in the whole fish and specific tissues like liver for example.

How Rainbow Trout is Used for In Vivo/In Vitro Chemical Testing

For over 50 years rainbow trout, a fresh, cold water species, has been a standard test species and readily used for chemical exposure tests for its belief as a sensitive aquatic species to chemicals. Rainbow trout has been used for chemical lab testing because of its commercial availability, popularity as a sport and aquaculture fish and ease of access from hatcheries. Rainbow trout therefore is well known in chemical lab testing to study physiological parameters in the whole living animal (in vivo testing).
Because of its reputation rainbow trout is also attractive to use for environmental in vivo and in vitro bioaccumulation metabolism studies for similar reasons. Studies using species other than rainbow trout from different habitats and adapted to different conditions are in demand to assess ecosystem and environmental risk. Studies of chemicals using different fish species are sparse. At the same time, regulatory agency protocols require use of proven, well-studied and the most readily available species to use for chemical lab testing even though they may not be the best fit to compare with other species including mammalian species. Faced with this dilemma, KJ Scientific is poised to find a better fit – from an array of different species.

KJ Scientific in vitro chemical lab testing – does not use the whole organism in vivo at once – but instead utilizes cells, tissues and tissue fractions from fewer animals to test how substances interact in the body. In vitro technology operates on a cellular and molecular level to predict bioaccumulation of chemicals in the environment. KJ Scientific uses the new Organisation for Economic Co-operation Development Test Guidelines (OECD TG) 319A and 319B in vitro metabolism technology and reduces the number of animals needed for chemical lab testing by nearly 100 percent! (see our Blog “We Significantly Reduce Animal Testing at Our Environmental Labs” on Dr. Karla Johanning’s (Founder of KJ Scientific) work developing in vitro technology of liver cells for testing chemicals in the environment.

Rainbow trout has earned a reputation as a relatively easy source of liver S9 subcellular fraction and cryopreserved hepatocytes for in vitro lab testing. Rainbow trout prepared liver S9 fraction and cryopreserved hepatocytes preserve well largely maintaining chemical metabolizing ability and are easily transported. Dependable rainbow trout liver S9 fraction and cryopreserved hepatocytes from certified trout strains are commercially available from KJ Scientific for in vitro metabolism chemical lab testing at your request. KJ Scientific also conducts in vitro chemical assays with rainbow trout and other fish species with commercial chemicals and pharmaceuticals.

The liver is the organ that has always been regarded as the main site of drug metabolism. Drugs that are metabolized are processed and broken down (biotransformed) into different chemicals by enzymes in the liver. Since liver cells contain all of the processes and enzymes for metabolism that a drug is likely to face using in vitro liver S9 fraction and hepatocytes is the most direct way to measure metabolism of drugs. Biotransformation is challenging because it may also create new chemicals and cause new chemical effects than the original parent compound. By chemical lab testing in vitro KJ Scientific can identify what these chemicals are and actually identify them using chromatography and mass spectrometry.

The way a drug is metabolized, and other chemicals produced have a bearing on whether the chemical has potential to be toxic to organisms. The rate and pathways of metabolism however does vary among different species. The ability to biotransform protects organisms including fish from toxic effects by converting chemicals to more water-soluble forms that are then easily excreted. The chemicals that are produced by fish as a result of metabolism can make a difference too. There is a chance the chemical products created can be dangerous and cause toxic or deleterious effects from those which are not themselves harmful. The pharmaceutical industry does make comparisons between species early in the drug development process. By using in vitro studies they are supposed to select out the most relevant animal species closest to humans for use in future safety studies. The U.S. FDA admits that for purposes of human safety more work needs to be done to identify similar species for humans concerning metabolism of pharmaceuticals.

The Role of Genes in Drug and Chemical Metabolism

Our genetics (our DNA) also play a role in how we respond and metabolize drugs and whether they are toxic. The genes responsible for metabolism that we inherit from our parents (one from each parent) map out the products, in this case the specific proteins or enzymes in the liver, that react with and metabolize chemicals and pharmaceuticals. The specific genes that encode these enzymes produce similar enzymes but can have more than one form and produce different outcomes in this case how they perform or metabolize drugs.

Due to genetic differences between individual people with different metabolism (enzymes) different reactions to certain drugs are experienced. People may experience decreased effectiveness of drugs and even increased toxicity because of their genotypes. Besides variability by individual, the enzymes that metabolize pharmaceuticals even differ among ethnic groups.

Different species of fish have comparable liver drug-metabolizing enzyme activities as higher vertebrate species. However, because of the chromosomal arrangement, additional gene duplication events during evolution, high diversity (over 30,000 different fish species) as well as various adaptive modes of reproduction, habitat use, and feeding behavior, fish have developed many more different forms (isoforms) of metabolic and other enzyme systems. Rainbow trout does have many of the same enzymes as well as the ability to metabolize pharmaceuticals as humans but there are differences. Some of the genes responsible for metabolism of pharmaceuticals in rainbow trout are similar in humans but the actual metabolism that occurs may vary. Consider the common human pain killer, ibuprofen as an example. Humans are capable of metabolizing ibuprofen though rainbow trout can metabolize some of the ibuprofen but not all.

Rainbow trout does appear to metabolize some drugs differently than humans therefore further genetic differences may be at play. Since genetics plays a significant role in determining the metabolism of drugs in humans the genetics of the fish species is also as important. Essentially how related fish are to humans is a most important factor when considering a representative species in chemical lab testing.

The Evolution of Fish Species

We have evolved and descended from fish; fish like rainbow trout are genetically like us too. Evolution is the process by which populations of organisms change overtime. Genetic variation is responsible for this change from gene mutation or gene recombination during cell division. This genetic variation is then passed on to the next generation and in this case from species to species. There is an evolutionary link that humans have descended from fish and have genetic similarities that control the development of the gills in fish and limbs of humans. Major organs like the liver and various body tissues are also found common. The freshwater zebrafish, a ray-finned fish species like rainbow trout, shares 70 percent of the same genes and 84 percent of human genes known to be associated with human disease for which pharmaceuticals are designed to combat.

There was a major evolutionary event (genome duplication) that took place 450 million years ago when tetrapods (4-limbed critters together with humans) began to evolve separately from ray-finned fish. More evolution then followed within tetrapods and ray-finned fish. Some genes were lost but some genes and gene function remained unchanged. What can be learned from this evolutionary relationship is that the fish most similar genetically to humans is the fish species most closely related to ray-finned fish. This is the fish species that descended just prior to that first major evolutionary split. Some living representatives of these fish species before the split are bowfin and gars. The genetic mapping of gar shows gar possess the same human genes that control the development of tissues and organs – brain, bone, heart, muscle. These human genes found in gar are ultimately responsible for human development and health importance. Because of this close genetic relationship of gar and humans Dr. Karla Johanning of KJ Scientific and her colleagues sees gar as potentially another representative fish species to use for in vitro metabolism for chemical risk assessment.

Comparison of In Vitro Metabolism of Different Fish Species

Pharmaceuticals and personal care products are widely found in our waters from wastewater treatment and therefore bioaccumulation of these chemicals in fish and other aquatic organisms is a regulatory concern. Determining whether a chemical bioaccumulates in an organism is an important part of environmental risk assessments. For the most part bioaccumulation is estimated using in vitro study and the data obtained is used to model metabolism. These estimates are directly influenced by the organism’s ability to metabolize (biotransform) a chemical or pharmaceutical.

To determine if rainbow trout can metabolize pharmaceuticals that humans could scientists from Baylor University looked at in vitro metabolism of 12 pharmaceuticals using rainbow trout liver S9 fractions. From this study they found that only 2 drugs (propranolol, diclofenac) of the 12 drugs could be metabolized by rainbow trout. They observed that propranolol was extensively metabolized, diclofenac was somewhat metabolized. Diphenhydramine is an example of one of the drugs that was not metabolized in rainbow trout. Overall therefore their results suggest that pharmaceuticals have a greater tendency to bioaccumulate in fish more than expected.

Dr. Karla Johanning and her colleagues decided to test those same pharmaceuticals in alligator gar. They felt compelled to investigate in vitro metabolism of some of these drugs in alligator gar and to compare it with reference rainbow trout. The pharmaceuticals propranolol, diclofenac, and diphenhydramine were chosen for chemical lab testing using liver S9 fraction preparation of both species. For reference, propranolol is a beta blocker used to treat high blood pressure, chest pain (angina), and uneven heartbeat. Diphenhydramine is an antihistamine used to relieve symptoms of allergy, hay fever, and the common cold. Diclofenac is a nonsteroidal anti-inflammatory used to treat pain, migraines and arthritis. Diclofenac was particularly chosen because of its history of increasing presence in U.S. southern inland waters, alligator gar habitat.

From the preliminary results of liver S9 fraction Karla finds that alligator gar is able to metabolize all the drugs extensively when compared to rainbow trout. Notably, diclofenac is metabolized significantly more in alligator gar liver S9 fraction than in rainbow trout. The genomics show that alligator gar is more genetically related to humans than rainbow trout, and Karla’s study indicates that alligator gar may metabolize drugs much more like a human, a mammalian species. In essence, ancient species such as alligator gar may be a better representative species for biomedical research than the ray finned fish, rainbow trout.

To emphasize, variation exists in metabolism between species that needs to be addressed when modeling bioaccumulation for chemical risk assessment. Obtaining chemical lab testing data for metabolism in vitro from other species supports environmental risk assessment efforts by providing a better prediction for bioaccumulation representative of more species. The OECD Test Guidelines 319A and 319B for performing in vitro toxicology metabolism studies, however, only address the use of trout liver cells when making predictions of bioaccumulation. From her preliminary research, Dr. Johanning sees that data from pharmaceutical and biomedical in vitro testing of more species adds needed value when predicting metabolism. Multi species data can be applied and integrated into the OECD TG to make them more robust and comprehensive. As you see, KJ Scientific is ready to contribute in this effort.

KJ Scientific is ahead in research conducting chemical studies comparing liver metabolism (using liver S9 fractions and cryopreserved hepatocytes) in rainbow trout and alligator gar and other fish species (brown trout, carp, bluegill, fathead minnow, largemouth bass, sturgeon and, bowfin), a reptile (American alligator) and mammalian species (human, rat, mouse and monkey).

KJ Scientific has the capacity to do chemical lab testing using in vitro liver S9 fraction and hepatocytes using your desired species of choice. Contact us for your specific testing needs and custom liver preparations.

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