C ONCLUSIONES

M

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Associate Professor of Toxicology, Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Denmark

14

● Thein vitro testing of metabolism is discussed.

● Thein vitro testing of acute toxicity is presented.

● Thein vitro testing of genotoxicity is described.

● Thein vitro testing of developmental toxicity is discussed.

antibodies) to the different isoenzymes; if the metab-olism is catalysed by one of the isoenzymes inhib-ited, the amount of metabolites will be decreased or nil (found by measuring the amount of metabolites after incubation). The microsomes can also be used to test if a compound can inhibit or reduce the activ-ity of the cytochrome P450 enzymes. This is done by incubating the microsomes with the test compound and substrates for the different enzymes. A reduc-tion in metabolism of one of these substrates indi-cates that the test compound inhibits the enzyme that catalyses that specific reaction.

Metabolism can also be tested by using either pri-mary cultures of hepatocytes or liver slices. This sys-tem has the advantage that it contains all phase I and phase II metabolizing enzymes, which makes it possi-ble to measure all the different metabolites. However, the activity of some of the cytochrome P450 enzymes in particular decreases very fast in vitro and therefore these systems are applicable only for short incubation periods. Hepatocyte cultures have another advantage

as they can be used to test if compounds induce or reduce the activity of enzymes and this can be esti-mated at the expression level. A promising new way to test metabolism is under development: genetically engineered cell lines that are capable of both phase I and phase II metabolism. The metabolism should preferably be catalysed by human enzymes.

14.3 Acute Toxicity Testing

General acute in vitro cytotoxicity can be estimated using different cell lines or primary cultures and different endpoints to estimate cell death and calcu-late the LC₅₀, i.e. the concentration of the test compound that kills 50% of the cells. Cells or cell lines are incubated with different concentrations of the test compound, both with and without the S9 fraction, in order to see if the test compound is metabolized into a more or less toxic compound.

After incubation the cell death can be measured in several ways. The integrity of the cell membrane Homogenization

Tissue (e.g. liver)

Homogenate

Centrifuge at 400g

Whole cells and nuclei in sediment

Centrifuge supernatant at 12,000g

Supernatant = S9

Sediment (pellet) = mitochondria

Centrifuge supernatant at 105,000g

Supernatant = cytosolic fraction Sediment = microsomal fraction

Resuspend pellet, centrifuge at 105,000g

Sediment = washed microsomal fraction

Fig. 14.1. Isolation of subcellular fractions from animal tissue. Adapted from XenoTech web page.¹

can be determined by measuring leakage from the cell (LDH assay, which measures leakage of lactate dehydrogenase into the medium), the exclusion of tryphan blue or the active uptake of neutral red.

The cell viability can also be estimated by measur-ing the mitochondrial metabolism (MTT assay, in which 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), a yellow tetrazole salt, is reduced to a purple formazan salt), or the synthesis of DNA and/or of protein. Lately apopto-sis assays have been used more extensively. None of these general cytotoxicity tests have been accepted as OECD guidelines but the results found in these tests in different cell lines using different endpoints can be used for screening and ranking compounds as a first approach to selecting and prioritizing them for further studies, i.e. selecting the least toxic candidates for new food additives, pesticides or drugs.

The tests may also give an indication of targets for the toxic effect and help to estimate the first dose in acute in vivo toxicity studies, as a certain

correlation has been found between the LC₅₀ and LD₅₀ doses. In this way a reduction in the number of animals used in the first in vivo studies has been possible.

In vitro toxicity tests that can replace the ‘Draize’

acute in vivo skin and eye irritation and corrosive test(s) have also been developed, some of which have been accepted as OECD guidelines while others have been accepted only in certain countries, as overviewed in Table 14.1. Examples of these tests include the ‘In vitro skin corrosion – human skin model test’, which uses an artificial human skin model, and the ‘Membrane barrier test method for corrosion’. The ‘Transcutal electrical resistance test (TER)’ is also a well-described method measur-ing the resistance over a cell membrane; a decrease in resistance is a measure for toxicity. The in vitro acute eye tests include tests on isolated bovine cor-nea, isolated chicken eyes and isolated rabbit eyes.

These are in vitro test methods that can be used to classify substances as ocular corrosives and severe irritants. The use of isolated corneas from the eyes

Table 14.1. Examples of corresponding in vivo and in vitro methods. Numbers in brackets are the OECD guideline numbers.

Toxicological test In vivo method In vitro method

Skin irritation/corrosion Acute dermal irritation/corrosion (404) Transcutal electrical resistance test (TER) (430)

Skin corrosion: human skin model test (430) Membrane barrier test method for skin corrosion (435)

Eye irritation/corrosion Acute eye irritation/corrosion (405) Embryonal chicken egg (HET-CAM)^a Isolated bovine cornea (BCOP)^a Isolated chicken eye (CEET)^a Isolated rabbit eye (IRE)^a Mutagenicity/genotoxicity Mammalian erythrocyte micronucleus

test (474)

Bacterial reverse mutation test (471)

Mammalian bone marrow chromosomal aberration (475)

Saccharomyces cerevisiae gene mutation assay (480)

Sex-linked recessive lethal test in Drosophila melanogaster (477)

Saccharomyces cerevisiae mitotic recombination assay (481) Rodent dominant lethal (478) Mammalian chromosome aberrations

assay (473)

Mouse spot test (484) Mammalian gene mutation test (476) Mouse heritable translocation assay (485) Micronucleus test (487)

Unscheduled DNA synthesis (UDS) with mammalian liver cells (486)

Unscheduled DNA synthesis (UDS) in mammalian cell cultures (482) Teratogenicity Prenatal developmental study (414) Whole embryo culture (WEC)^a

Micromass test (MM)^a Embryo stem cell test (EST)^a

aThese in vitro tests have not (yet) obtained regulatory acceptance from OECD.

of cattle slaughtered for commercial purposes thus avoids the use of laboratory animals.

As chemical compounds may be activated by light, the ‘In vitro 3T3 NRU phototoxicity test’

was developed. This method measures photo-cytotoxicity by the relative reduction in viability of cells as measured using the uptake of neutral red, a dye that is actively transported into the cells. In order to test the activation of the compound, cells are incubated with the compound in the absence versus presence of a non-toxic dose of UV light.

14.4 Mutagenicity/Genotoxicity Testing Specific concerns about the possibility of a com-pound or product causing mutations (changes) in the genetic material have made these tests the most intensively used. The first tests were developed at the beginning of the 1970s by Bruce Ames and they have proved to be very useful indeed, especially as they are both cheap and quick instruments for the screening of, for example, a battery of synthesized candidate compounds in the development of a new food additive, pesticide or drug. Positive results (i.e. the compound induces mutations) will most often lead to termination of the further planned programme for the compound in question. In contrast, a negative result has to be confirmed in other in vitro tests using mammalian cell lines and then the compound might be tested in one or more in vivo assays using higher animals, typically mammals in the form of a rodent species.

Within the bacterial assays for mutagenesis the ‘Bacterial reverse mutation test’ uses amino acid-requiring strains of either Salmonella typh-imurium (the Ames test) or Escherichia coli to detect point mutations by base substitutions or frameshifts. Thus, the test uses amino acid-dependent strains of S. typhimurium and E. coli, which, in the absence of an external amino acid source, cannot grow to form colonies. However, colony growth is resumed if a reversion of the mutation occurs due to exposure to a mutagen, allowing the production of the amino acid. Spontaneous reversions occur within each of the strains, but mutagenic com-pounds included in the growth medium cause an increase in the number of revertant colonies rela-tive to the background level. Bacterial culture medium is inoculated with the appropriate S. typh-imurium or E. coli strain and incubated overnight.

Different concentrations of the test compound are mixed with the bacteria in individual test-tubes and

the S9 fraction is added to half of them. The test-tube contents are spread on agar plates without the amino acid and incubated for 48–72 h. Often a dose range-finding test for the compound under investigation is carried out using the S. typhimu-rium strain TA100 over a wide dose range. The increase in colony numbers as a function of the test compound concentration indicates how mutagenic the compound is.

The addition of the S9 fraction – an external phase I metabolizing system – will give an indica-tion about the metabolites of the compound, i.e. if they are more or less mutagenic than the parent compound. Similar gene mutation assays have been developed using yeast cells (Saccharomyces cerevisiae) and mammalian cell lines.

If mutations (microlesions) have been induced by chemicals, UV light or virus infections, the cells will start repairing the mutations. This process has been utilized in two different mutagenicity tests:

the UDS (‘Unscheduled DNA synthesis test’) and the COMET assay. The latter has not yet been accepted by OECD, but is being used increasingly.

In the UDS test cells are exposed to different con-centrations of the test compound, again with and without the S9 fraction added. Unscheduled DNA synthesis, i.e. DNA synthesis that is not initiated as part of cell proliferation, can be estimated by add-ing a radioactive nucleotide and by measuradd-ing the radioactivity of the cells. The unscheduled DNA synthesis can be estimated and thereby the muta-genicity of the test compound.

In vitro mammalian chromosome aberration test

The purpose of the ‘In vitro chromosome aberra-tion test’ is to identify agents that cause structural chromosome aberrations in cultured mammalian somatic cells. Structural aberrations (macrolesions) include any numerical or structural change in the usual chromosome complement (the whole set of chromosomes for the species) in a cell or organism.

In humans, the chromosome complement (or kary-otype) consists of 46 chromosomes. An example of a structural change includes the gain, loss or rear-rangement of chromosomal segments caused by the chemical compound (Fig. 14.2). Another is the formation of ring chromosomes. When one break occurs in each arm of a chromosome, the broken ends of the internal centromeric fragment may join, resulting in the formation of a stable ring chromosome.

Each of the two end segments lacks a centromere. Such acentric fragments are lost during cell division because the centromeres are essential in the cell division process, being the location for binding of the spindles.

The ‘Micronucleus test’ is a test that can be used both in vivo and in vitro. The test detects chemi-cally induced chromosomal damage determined by the increased frequency of micronucleated poly-chromatic cells. The assay is sensitive to both clas-togenic compounds and compounds that interfere with the spindles. Micronuclei are cytospasmic chro-matin masses from chromosomes or chromosomal fragments that are not incorporated into daughter cells during mitosis (Fig. 14.3).

14.5 Developmental Toxicity Testing The effort to find alternative in vitro developmen-tal tests has been increasing as the in vivo develop-mental toxicity tests – in particular the two generation test – require by far the largest propor-tion of animals in safety studies as described by REACH. Three in vitro methods, the ‘Embryonic stem cell test’ (EST), the ‘Micromass test’ (MM) and the ‘Rat post-implantation whole embryo cul-ture test’ (WEC), have been validated and are rec-ommended as screening tests for developmental toxicity (Table 14.1). However, no OECD guide-lines have yet been made. These tests cover only certain aspects of developmental toxicity and mainly measure embryotoxicity as an endpoint.

In the EST assay embryonic stem cells that can be maintained in the undifferentiated stage are exposed to the test compound. The inhibition of cellular dif-ferentiation mainly into cardia myoblasts is meas-ured to estimate the embryotoxicity of the compound.

In the MM assay cultured chick embryo limb cells are exposed to the test compound and toxicity is measured with a focus on the differentiation of limb bud cells into cartilage-producing chondrocytes. The WEC utilizes rat embryo cultures on day 9.5 of ges-tation. During 48 h of culture major aspects of orga-nogenesis occur, such as heart development, closure of the neural tube and development of ear, eye, limb buds and brachial bars. Exposure to chemicals at this time can lead to retardation of growth and malformation of one or several of the organs under development. These malformations are evaluated using a morphological scoring system. This test and the EST have shown excellent predictivity and preci-sion for strongly embryotoxic compounds (100%), and for non-embryotoxic compounds the precision was 65 and 72%, respectively. However, these tests have only been used to test compounds with a lim-ited number of toxicological mechanisms, and an external metabolizing system such as the S9 fraction has not been applied.

14.6 Toxicogenomics – Proteomics In recent years toxicogenomics and proteomics have increasingly been used to study the mechanisms of toxic effects caused by exposure to chemical com-pounds. In toxicogenomics the expression or change

dicentric dicentric

fragment

fragment ring

Fig. 14.2. A spread of chromosomes showing aberrations such as dicentric chromosomes and chromosomes without centromeres plus a ring chromosome. With permission from the National Institute of Radiological Sciences, Japan.²

micronucleus

Fig. 14.3. A micronucleus detected by Geimsa stain-ing. With permission from the ‘Micronucleus test’ web page.³

in expression of genes is analysed by use of cDNA microarrays or by quantitative PCR (qPCR). Such changes in gene expression will cause a change in protein level in the cell. These changes can be analysed by using proteomics to isolate the proteins and running a two-dimensional polyacrylamide gel electrophoresis. By doing this it is possible to distinguish all the proteins and find the changes. These methods are often used to identify biomarkers for the toxic effect. Such biomarkers found in vitro using different cell lines can afterwards be used in vivo.

14.7 Conclusion

Over the last three decades the area of in vitro toxico-logical methods has progressed tremendously, owing to the development in knowledge of cell biology and cell and tissue culture methodologies. Nevertheless there are still some drawbacks including the loss of tissue-specific functions in many in vitro systems, such as the loss of metabolic activity in hepatocyte cultures. Also the tissue–tissue interaction is lacking in the in vitro assays. Furthermore, the in vivo bio-availability of the test compound has to be considered because, if its intake is oral, then the compound first of all has to be absorbed in the gastrointestinal tract and then distributed to the different organs. In addition the compound may be accumulated in certain tissues in vivo, e.g. adipose tissue. On the other hand, the use of in vitro methods reduces the number of animals used in the initial phase of toxicity testing, and elucidation especially of the toxicological mechanisms and the kinetic studies permitted by in vitro systems will have regulatory impact.

Notes

1 XenoTech (not dated) Subcellular Fractions. XenoTech LLC, Lenexa, Kansas; available at http://www.

xenotechllc.com/Products/Subcellular-Fractions

2 NIRS (2007) Inevitable aspect to radiation emergency medicine. National Institute of Radiological Sciences, Chiba, Japan; available at http://www.nirs.go.jp/ENG/

research/radiation_emergency/04.shtml

3 Vrije Universiteit Brussels (not dated) Micronucleus test. Laboratory Cell Genetics, Vrije Universiteit Brussels, Belgium; available at http://we.vub.ac.be/~

cege/volders/ENG/tests/MN.htm

Further Reading

Attenwill, C.K., Goldfarb, P. and Purcell, W. (eds) (2000) Approaches to High Throughput Toxicity Screening.

Taylor & Francis, London.

Eisenbrand, G., Pool-Zobel, B., Baker, V., Balls, M., Blaauboer, B.J., Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J.C., Pieters, R. and Kleiner, J. (2002) Methods of in vitro toxicology. Food and Chemical Toxicology 40, 193–236.

Gad, S.C. (2000) In Vitro Toxicology. Taylor & Francis, London.

Gad, S.C. (2002) Drug Safety Evaluation. John Wiley &

Sons, New York, New York.

O’Hare, S. and Atterwill, C.K. (eds) (1995) In Vitro Toxicity Testing Protocols. Methods in Molecular Biology, vol. 43. Humana Press, Totowa, New Jersey.

Stacey, G.N., Doyle, A. and Ferro, M. (eds) (2001) Cell Culture Methods for In Vitro Toxicology. Kluwer Academic Publishers, Norwell, Massachusetts.

Tiffany-Castiglioni, E. (ed.) (2004) In Vitro Neurotoxicol-ogy: Principles and Challenges. Methods in Pharma-cology and ToxiPharma-cology. Humana Press, Totowa, New Jersey.

15.1 Primary and Secondary Metabolites as Toxicants

The plant kingdom (Plantae) comprises a number of groups with an amazing range of diverse forms.

This kingdom is second in size only to the arthro-pods. Plantae include the water-living algae, which range from single-cell organisms to seaweeds more than 10 m long, and a number of terrestrial groups of so-called spore plants, i.e. the mosses, plants belonging to the families Lycopodiaceae (e.g. club moss or stag’s horn) and Equisetaceae (e.g. horse-tail), respectively, and the ferns. In addition the plant kingdom includes the seed plants (Spermatophyta) made up of the gymnosperms (the cycads, Ginkgo biloba (the maidenhair tree) and the conifers) and the angiosperms (the flowering plants), which are subdivided into the monocotyle-donous (monocots) and the dicotylemonocotyle-donous (dicots) plants. The first of these two groups includes all of the grasses – and hence the cereals – together with palms, lilies (including onions) and tulips, just to give some examples. Of food-relevant plants the dicots include the fruit trees, all of the cabbages, etc. An overview of the plant kingdom (Plantae) according to the scientific understanding used here (other systems exist) is given in Table 15.1.

Plants were shown early in the 20th century to contain a high number of so-called secondary metabolites, i.e. low-molecular-weight organic compounds biosynthesized by the plant but not essential to its growth and survival if isolated from interactions with other organisms. Such com-pounds typically are thought to be protective to the

plant by deterring herbivorous organisms from eating it by being bad tasting or toxic, or both.

From the point of view of the mechanisms of deterring animals from eating the plant, we often divide the secondary compounds into anti-nutritional and toxic compounds:

● toxic (poisonous) compounds destroy life or impair health by their own action or by the action of their metabolites formed (in the body) after uptake; and

● anti-nutritional compounds impair health by destroying nutrients/vitamins or by reducing the uptake of such essential compounds (by different mechanisms).

Macromolecular compounds such as polynucleic acids (DNA, RNA), proteins and polysaccharides are seldom toxic, and especially not upon inges-tion. In saying this maybe we should just mention that we thereby exclude the elicitation of an aller-genic response by an ingested protein as a toxic effect. Also it should be remembered that the harm-ful effects potentially caused by DNA/RNA in the form of viruses and proteins in the form of prions are not toxic effects.

In spite of what has just been stated, a few indi-vidual proteins and classes of protein still have the ability to cause toxic or anti-nutritional effects in higher animals including man. Thus, the plant pro-teins ricin and abrin are highly toxic, while the two groups of proteins called lectins and proteinase inhibitors possess anti-nutritional effects. The enzyme thiaminase, which degrades the B vitamin

In spite of what has just been stated, a few indi-vidual proteins and classes of protein still have the ability to cause toxic or anti-nutritional effects in higher animals including man. Thus, the plant pro-teins ricin and abrin are highly toxic, while the two groups of proteins called lectins and proteinase inhibitors possess anti-nutritional effects. The enzyme thiaminase, which degrades the B vitamin