in vitro toxicity testing protocols

The use of lower organisms not protected by legislation, including invertebrates, plants, and microbes.a The use of embryonic and larval vertebrates before they reach the developmental

CHAPTER 1

Chris K Atterwill

1 Introduction The safety assessment of new chemicals and pharmaceuticals and the incorporation of these data into a human risk assessment package requires

a large number of expensive, regulated tests in animal species including,

in some cases, nonhuman primates (I-3)

There are currently a wide range of animal replacement alternative opportunities in industrial chemical and drug development (Table 1) (4,.5) Although in vitro methodology has long been used as a basic labo- ratory tool for defining biological and toxicological processes in differ- ent cellular systems, application and use alternatives in industrial compound discovery (i.e., research and development) is slow Coupled with a relatively low innovation rate in the design of new in vivo tests for the toxicological and safety evaluation of new compounds, this has both ethical and resource implications

From a basic scientific viewpoint in vitro toxicological models have made important contributions in elucidating, e.g., the cellular and molec- ular mechanisms involved in apoptotic and necrotic cell death and in carcinogenesis and the role of mediators, such as free radicals and oncogenes, in these processes (6)

The “take-up” of in vitro systems in toxicity testing is, however, now gradually occurring in industry and resources are being invested slowly into the area for both ethical and scientific reasons A lot of emphasis currently lies on the ethical question as public sensitivity to animal use in safety testing increases This has occurred most significantly in the case

From Methods m Molecular Wology, Vol 43 In Wtro Tox/c/ty Testmg Protocols

Edited by S O’Hare and C K Atterwlll Copyright Humana Press Inc , Totowa, NJ

1

Table 1 Ammal Replacement Alternatives Improved storage, exchange, and use of information, so that unnecessary repetition of experiments on animals could be avoided

Maximum use of predictions based on physical and chemical properties of molecules Mathematical modeling of quantitative structure-activity relationships

Molecular modeling and the use of computer graphics

Mathematical modeling of biochemical, physiological, pharmacological, and toxlco- logical systems and processes

The use of lower organisms not protected by legislation, including invertebrates, plants, and microbes.a

The use of embryonic and larval vertebrates before they reach the developmental stage

at which time they become protected by law a

The use of in vitro methods including subcellular fractions, perfused organs, tissue shces, and cell suspensions; and cell organotypic cultures *

Human studies, including epidemiology, postmarketing surveillance, and the properly regulated use of volunteers.a

% vitro areas

of cosmetic and toiletry safety assessment and the safety testing of chemi- cal intermediates used in drug synthesis, which have classically used the controversial Draize, guinea pig, and rabbit tests, and for which valid alternatives now exist Furthermore, in drug development the classic LD,, tests for acute toxicity have not only been partially replaced by the “fixed dose” procedure, but much work is being carried out by Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) on new and rapid in vitro predictors for acute cytotoxicity using human cell lines

Utilizing Alternative Test Models The development and registration of both new drugs and chemicals currently requires the submission of a large battery of in vivo toxicity data derived from a number of species for the “risk-assessment” process (1-3) The compilation and validation of the animal batteries has been largely empirical over the years and, although being fairly well-proven for detecting toxic phenomena in animal species, can have limited pre- dictive value for human safety assessment for some of the reasons listed

in Table 2 (a,b) There are large lists of chemicals with good animal- human toxic correlations, but equally, lists of compounds exist that have been withdrawn from the market because of the increasing number of clinically reported adverse reactions (ADRs)

Alternative Method of Assessing Toxicity 3

Table 2a Reasons for Incorrect Predictions from Animal Toxicity Studies

False negative responses

Effect not looked for

Use of inappropriate assay methoda

Improper timing of assay

Insufficient target organ exposure0

Incorrect evaluation of an experimental finding0

Failure to consider absence of preexisting pathological condition

Inability to identify and measure adverse effect0

False negative and false positive responses

Failure to consider differences in metabolic activation and detoxrfrcationa

Disregard of anatomrcal and physiological differences between species“

Inability of animals to express human-specific reaction pattern9

Table 2b Conversion Factors for Predictmg from Animal Studies to Individuals at Risk

Receptor sensitivitya People with infections Anatomical characteristics Immunosuppressed individuals Physiological characteristicsa Alcohol or drug abusers

Species-specific responsea Patients with organic disease Mechanisms of actiona Occupationally exposed indivrduals

OAreas in which alternatlves tests can have an impact (Data taken from table compiled by G Zbinden, personal communication.)

It is also well accepted that during the development of a “safe” and effective pharmaceutical (or agrochemical agent) there is a large attrition rate throughout the safety assessment process with massive financial implications When one superimposes on this the varying worldwide regulatory requirements for administration of a new compound to humans, one can see a number of important reasons for developing in vitro toxicity testing systems either as prescreens or as adjuncts to cur- rent in vivo test packages This, together with harmonization of the cur-

rent regulatory requirements, will hopefully improve the sensitivity and specificity of animal tests (3)

3 Summary of Gains

1 Financial gains: Reduce attrition rate by use of prescreening strategies prior

to full regulatory animal study packages and develop better predictors of human toxic phenomena

2 Scientific gains: Describe more effectively the lesions seen m vivo from regulatory studies and give better definition of safe concentration and clinical dose Define “direct toxicant effects” on target organs as opposed

to indirect effects, and give human reaction mdication using primate cells “Adjunct” studies will improve sensitivity and specificity of ani- mal studies

3 Ethical gains: Implementation of 3R strategy (reduce, refine, replace am- ma1 testing) Supplement and reduce current m vivo toxicity tests, particu- larly those involving distressing procedures and the use of large mammals and primates

4 Principles, Aims, and Types of In Vitro Toxicity Testing

The general advantages and disadvantages of in vitro testing for toxic-

ity are described in Table 3

5 Validation The successful use and industrial and regulatory acceptance of a new

in vitro test model depends on a certain degree of validation (7,s) Detailed validation is generally required if the in vitro test is to replace an in vivo test or is to be used as a prescreen where financial factors are critical However, when the result from an in vitro procedure is submitted to a regulatory authority along with that from an in vivo test package in order

to explain a lesion, then full validation is not formally necessary as long

as good laboratory practice (GLP) procedures have been adopted in the execution of that test and the test and endpoints have acceptable rele- vance For example, the gradual replacement of the Draize procedure by tests such as the EYETEX or SKIN2 tests has required extensive valida- tion of that test So, for example, would the use of a prescreen for a new immunosuppressant with adrenal toxicity where there were a limited number of available backup compounds or they were very expensive to synthesize On the other hand, if a drug company were trying to confirm

to the Food and Drug Administration (FDA) that a particular drug had no

Alternative Method of Assessing Toxicity 5

Table 3 Advantages and Disadvantages of In Vitro Systems for Detection

of Xenobiotic-Mediated Toxicity Advantages (general)

Detect direct (vs Indirect) toxrc/cytotoxic effects on target organ

Use controlled conditions of exposure-concentration of toxrcant known

Study parent compound vs metabolite (rt liver S9 metabolizing fractions from different species)

Study effects on cells vs subcellular organelles

Has resource implications (time, animals, number of compounds tested)

Disadvantages (general)

Systems not always representative of mature, differentiated target organs (cells dedifferentiated in cell lines?)

Xenobiotic concentrations not representative of those in vivo (e.g., plasma

protein bindmg factors)

Biological barriers absent (e.g., blood-brain barrier in neural cultures of CNS) Metabolite profiles differ

Difficulty of culturing/maintaining certain target organs in vitro

direct neurotoxic effect in humans despite some minor behavioral changes detected in the rat, then submission of data from cultured human exposed neurons would be acceptable, probably without full validation

of that particular culture model

Accepted validation criteria for an in vitro system are described in Table 4 and include definition of the specificity, sensitivity, and predic- tive value of such a test The validation parameters are obtained by con- ducting blind validation trials It is my belief that the requirements for in vitro test validation can be summarized as follows:

1 Full validation involving multicenter coordination: To support in vrvo test replacement

2 In-house validation: In vivo test reduction, to support, e.g., development

of a prescreen,

3 Limited inter- or intralaboratory: To support refinement or supplementa- tion of in vivo toxicity test data, to develop adjunct tests; use of the model

to define basic scientific toxic phenomena

It is believed that validation should not be used as an excuse for nonadoption, nondevelopment, or nonacceptance of in vitro methodol- ogy Sadly, and largely for political reasons, this scenario still exists in many companies and countries

Table 4 Validation Criteria for In Vitro Test Models

A formal validation study will require:

Careful selection of chemicals (mimmum 20-40?)

Use of chemical pairs

Toxicological classification from m vivo data

“Blind” testing to be performed

Method for evaluation of test outcome (absolute values)

Method for evaluation of test performance

Methods of expressing test performance

Other points

Are there good in vivo comparative data for compounds chosen?

Which kind of in vivo assay trying to emulate/evaluate in vitro?

Agree with collaborating centers in validation trial at beginning who will be organizing and collatmg data

6 Spectrum of Available In Vitro Toxicity Tests

The currently available models in In Vitro Toxicology (Table 5) (5-8) span six main areas: reproductive toxicity, mutagenicity, irritancy test- ing, immunotoxicity, target organ toxicity (including endocrine and neu- rotoxicity), and ecotoxicity involving the use of fish, invertebrates, and

so on Within these main areas there are also important subareas

As alluded to above there are various modes in which to operate these tests in an industrial setting and generally the mode predominance varies significantly according to both scientific area and whether or not one is operating in the drug, chemical, or cosmetic industry For example, a test system might progress from unvalidated use in the fine description of a pathologically identified lesion for a lead development compound, to the subsequent, semivalidated use of this system in a prescreening mode for second-generation drug candidate compounds Alternatively, the agro- chemical industry has developed a tiered in viva/in vitro hierarchical model for the labeling of industrial chemicals as skin irritants This latter development was performed under the auspices of the British Toxico- logical Society, showing how the scientific and industrial communities can interact so well on such issues Here, a chemical for irritancy classi- fication would proceed from tests on isolated skin or cells in vitro to tests

in a limited number of animals in vivo depending on negative or positive outcomes in the initial in vitro tests

Alternative Method of Assessing Toxicity 7

Table 5

In Vttro Models Currently Available in Toxicology Mutagenicity testing

Irritancy testing Reproductive toxicity testing Quality-Structure Activity Relationship (QSAR) Target organ toxicity Immunotoxicity Hemtc system Endocrine toxicity Neurotoxtctty Acutelcytotoxicity testing

7 Recent Successes and Developments in In Vitro Toxicity Testing

It is refreshing to observe the momentum that is now gathering in this field (see Table 6) and the way in which “in the face of adversity” some tests are being accepted as full replacement alternatives It is noteworthy

to say that a lot of this energy has been provided by the public and by academic research centers

Apart from the mutagenicity test area where many innovations con- tinue to occur, some of the following recent developments in other areas warrant attention

1 Eyetex, Skmtex, and Corrosrtex tests for eye and skin irritancy (Ropak Corporation Ltd) and the SKIN2 Model (Advanced Tissue Sciences) More

recently, the Ropak Solatex system for predicting photoirritation in vitro

2 The use of hepatocyte “couplets” for in vitro investigation of xenobiotic

effects on bile flow Together with measurements of hepatotoxicity and fatty acid accumulation by these cells, rt may now be possible to obtain a

complete hepatoxicological profile in one in vitro model

3 Luminescent bacteria (Microtox test) for measuring the ecotoxic potential

of industrial effluent

4 More sensitive in vitro toxicity measurements using, for example, the mito- chondrial MTT test for succurate dehydrogenase activity This test gives a more sensitive and earlier prediction of toxicity than classical LDH or neu- tral red measurements

Table 6 Orgamzatlons Involved m the Development of Alternative Testing

Bodies for promotion of alternatlve nonanimal testing

FRAME-Fund for Replacement of Animals in Medical Experiments

ERGAT-European Research Group for Alternative Testing

EURONICHE-European group for alternatlve methods for biology teaching CAAT-Center for Alternatives to Animal Testings (Johns Hopkins Medical School, Baltimore, MD)

Dr Hadwen Trust-Nonammal research and testing strategies (UK-based)

Societies, conferences, and journals advancing alternative testing

PIVT-Practical In Vitro Toxicology conference

IVTS-In Vitro Toxicology Society (UK)

Scandinavian Cellular Toxicology Society

FRAME Toxicity Committee and Conference

TIV-Toxicology In Vitro, Journal

ATLA-Alternatwes to Laboratory Animals, Journal

Hildegard Doerenkamp and Gerhard Zbinden Foundation for Reahstlc Animal Protection and Scientific Research, Switzerland

5 Measurements of calcium accumulation in single cultured neurons for the measurement of neurotoxicity

6 Tlered tests involving both simple and organotyplc organ systems; hierar- chical proceeds involving a battery of m vitro and in vivo models

8 Conclusions Industry and academia have come far in developing in vitro alterna- tives, and bodies such as Fund for Replacement of Animals in Medical Experiments (FRAME), European Centre for Validation in Alternative Methods (ECVAM), and Center for Alternatives to Animal Testing (CAAT) (USA), have simultaneously enhanced public and regulatory awareness (Table 6) The regulatory and industrial acceptance of new alternative tests depends on proper, well-coordinated validation trials at

a level befitting the intended use of the alternative test This has started through FRAME and European Community (EC) initiatives and good examples have been set by the cosmetics and toiletry industries More commercial “takeup” is still required for these new tests at the toxico- logical prescreening and in vivo adjunct testing level Regulatory har- monization of in vivo animal testing is occurring for both ethical and resource reasons The gradual replacement of the LD,, test by the “Fixed

Alternative Method of Assessing Toxicity 9

Dose” procedure for acute toxicity testing and the realization that 6 mo chronic testing (I) is sufficient to identify the most important pathology (excluding carcinogenicity) are most welcome changes The potential risks for humans in adopting alternative toxicity tests are few and ben- efits great if the data generated is used correctly The imminent replace- ment of all in vivo tests is unlikely but in the future may gradually occur

In the meantime in vitro tests will continue to supplement the somewhat

“impirical” animal tests for human toxicity

References

1 Volans, G N., Sims, J., Sullivan, F M., and Turner, P (eds.) (1989) Basic Science

in Toxzcology, V International Congress of Toxicology (ICTV), Taylor & Francis

2 Poole, A and Leslie, G B , eds (1989) A Practical Approach to Toxological

3 Lumley, C., Parkmson, C, and Walker, S R (1992) An international appraisal of the minimum duration of chronic animal toxicity studies Hum Exp Toxicol 11,

155-162

4 Parish, W E and Hard, G C (eds.), Toxicology In Vitro Proceedings of Second International Conference on Practical In Vitro Toxicology 4/5

5 Atterwill, C K and Steele, C E., eds (1987) In-Vitro Methods in Toxicology,

Cambridge University Press, Cambridge

6 Walum, E., Stenberg, K., and Jenssen, D (1990) Understanding Cell Toxicology-

7 OECD Environment Monograph No 36 Scientific Criteria for Validation of In

8 FRAME 21st Anniversary Issue (1990) ATLA, 18

LLC-RKl cells are maintained in culture and exposed to varying con- centrations of test compounds, The cultures are incubated for 48 h The cultures are then rinsed and incubated for 3 h in medium containing Neutral Red that is taken up by viable cells After rinsing, the dye present

in the cell population is liberated and the amount is quantified using a spectrophotometer, in order to obtain an indication of cell number Com- parison of the number of cells in control and test cultures provides an index of cytotoxicity and an indication of potential nephrotoxicity in vivo The maintenance and culture of a cell line such as LLC-RKl cells

is a relatively simple and inexpensive technique Additionally, LLC-RKl cells exhibit many features in common with kidney cells in vivo Among these is the unidirectional transport of solutes via the Na+K+ ATPase

From Methods m Molecular Biology, Vol 43 In Wtro Tox/c/ty Testing Protocols

Ed&d by S O’Hare and C K Atterwtll Copyright Humana Press Inc , Totowa, NJ

11

system As a result of this, one-way transport “blisters” are formed in the monolayer, a feature in common with primary kidney cells and other cell lines in culture (2) The application of such cultures to determine nephro- toxicity may potentially allow the rapid, highly reproducible testing of many chemicals on a routine basis

There are, however, disadvantages associated with using a cell line in culture The cells grow rapidly and are nondifferentiating Additionally, the cells in culture inevitably lose many characteristics of those in vivo

In particular, the loss of xenobiotic metabolizing activity may affect the sensitivity of the cells to certain chemicals and may raise concern when trying to directly extrapolate results to the in vivo situation

1.1 Neutral Red Uptake Assay Neutral Red is preferentially taken up into the lysosomes/endosomes

of the cell Absorbances obtained using the Neutral Red assays have been shown to correlate linearly with cell number over the specific optical density range obtained using this method Any chemical having a local- ized effect on the lysosomes/endosomes will, therefore, result in an arti- ficially low (or possibly high) reflection of cell viability and cell number This factor does, however, make the system useful to detect other chemi- cals that selectively affect the lysosomes, especially when it is used in conjunction with other tests capable of determining cell number (3)

2 Materials 2.1 Equipment

1 37°C incubator, hurmdified, 5% CO,/95% air

2 75 cm2 tissue culture flasks

3 24-well tissue culture plates

4 Inverted phase contrast microscope

5 Spectrophotometer

6 Hemocytometer

2.2 Reagents

1 Cell line rabbit kidney LLC-RKl cells

2 Dulbecco’s formulation tablets, without magnesium and calcium

3 PBS Trypsin/EDTA, 1X cone (dispense as 10 rnL aliquots into universals and store at -2O’C), Glbco Europe Ltd (Paisley, Scotland)

4 Eagle’s minimum essential medium (MEM) supplemented with 1% peni- cillin/streptomycin, 5% fetal calf serum N.B Omit penicillin and strepto- mycin if the test compound is an antibiotic

LLC-RKl Cell Screening Test

5 10,000 U/mL penicillm/l0,000 pg/mL streptomycin solution in saline Neutral Red stock solution; 100 mL 0.4% w/v Neutral Red in distilled water, filter sterilized Store at 4°C until required

6 Neutral Red medium: dilute the stock dye solutron (1 in SO) just prior to use with culture medium to give a final concentration of 50 pg/rnL

13 Neutral Red wash solution: 10% CaC12 in formaldehyde

14 Neutral Red resorb solutron: 1% glacial acetic acid, 50% ethanol, 49% distilled water

15 Test compounds: These should be drssolved in sterile water, ethanol, methanol, or drmethylsulfoxrde (DMSO), as approprrate at loo-fold the required final concentratron The final solvent concentration should be kept

at a constant level of 1% in the culture medium

3 Methods 3.1 Cell Maintenance

3.1.1 Preparation of Cells for Freezing

1 Count the cells and dilute to l/O.9 of the intended final concentration of l-2 x 106/mL in complete culture medium

2 Add DMSO to a final concentration of 10% to the cell suspensron immediately prior to adding to the vials

3 Aliquot 1.8 mL of cell suspension per vial and freeze at a rate of l”C/min

in liquid nitrogen,

3.1.2 Thawing and Culture of Cells

1 When required, thaw the cells rapidly in a 37°C water bath to avoid dam- age owing to the high DMSO concentration

2 Transfer immediately to a 75 cm2 tissue culture flask containing -30 mL medium (i.e., l-2 x lo6 cells/flask)

3 After 24 h, rinse the culture with 5-10 mL of PBS at 37OC

4 Add -30 mL fresh medium

5 Subculture the cells 2-3 times following thawing before using for test purposes

3.1.3 Subculture of Cells

1 When the cultures approach confluence remove the cells from the dish by trypsinization (N.B If the cells are not subcultured or used for test pur- poses, the medium should be changed every 3-4 d.)

2 Decant the medium and rinse the cultures with 5-10 mL of PBS at 37OC

3 Add 10 mL trypsin/EDTA (37°C) and incubate at 37°C

4 Remove the flasks after 20-30 s and examine visually to ensure the cells have begun to detach (i.e., round up)

5 Discard the trypsin/EDTA solution and return the flask to 37°C

6 After a further 1 mm, examine the cells and if necessary tap the side of the flask to aid detachment

7 Add 10 mL of complete medium to neutrahze the trypsin activity and spht

or use for test purposes

3.2 Test Procedure

1 After growing up the cells and preparing a cell suspension as described above, remove an aliquot of suspension and count the number of cells using

a hemocytometer and dilute to a concentration of lo5 cells/ml medium

2 Add 1 mL of the diluted suspension to all the wells of a 24-well plate Incubate overnight to allow adherence and recovery from the trypsm exposure

2 Remove growth medium and replace with 1 mL of each chemical dilution

in the appropriate wells in 24-well plates

3 Shake the plates gently to ensure an even distribution Incubate for 48 h

at 37°C

4 Remove the medium and determine the cell number by the Neutral Red assay (see Note 3)

5 From the preliminary results select six concentrations, spanning the range

of O-100% cell death, for an accurate determination of cytotoxicity

3.2.2 Determination of IDzO, ID,, and IDgO

1, Test each chemical concentration m triplicate on three separate occasions

2 Prepare:

a The appropriate solvent controls

b Six concentrations of the test chemical

LLC-RKl Cell Screening Test 15

3 Prepare the 24-well plates as before

4 After overmght incubation, remove growth medium and replace with 1

mL of the test chemical or the control to random wells (thus minimizing bias), but ensuring that a careful note is made of the treatment received by the cells in each well Shake the plates gently to ensure even distribution Incubate for 48 h

5 Estimate the cell number using the Neutral Red assay

3.2.3 Neutral Red Assay (1)

1 After 48 h, remove the medium from all the wells Wash gently with PBS Add 1 mL of Neutral Red medium per well Incubate for 3 h at 37OC, 5% CO2 in a humidified atmosphere

2 After 3 h, remove the Neutral Red medium Wash quickly with the Neutral Red wash solution Add 1 mL of resorb solution to each well Agitate the plates intermittently for a period of 15-20 min Transfer the solutions to cuvets and measure their absorbance at 540 nm using the resorb solution as

a blank

3.2.4 Results

1 Determine the mean value for the absorbance of the control cultures and adjust all indivtdual absorbances accordingly Mean the values for each treatment group and plot graphically Determine the ID,,, ID,,, and ID,, values from the curve

2 Mean the ID values from three separate experiments and give the final concentrations expressed as pg/mL or mmol/L Rank the chemicals for toxicity using the ID,, value (the section of the curve most likely to be linear and subject to least variation)

4 Notes

1 Volatile chemicals tend to evaporate under the conditions of the test, thus the ID,, value may be variable, especially when the toxicity of the compound is fairly low Chemicals that are unstable or explosive in water are also difficult to test (4J) Neutral Red is preferentially taken up into the lysosomes/endosomes of the cell Absorbances obtained using the Neutral Red assays have been shown to correlate linearly with cell number over the specific optical density range obtained using this method Any chemical having a localized effect on the lysosomes/endosomes will, therefore, result

in an artificially low (or possibly high) reflection of cell viability and cell number This factor does, however, make the system useful to detect other chemicals that selectively affect the lysosomes, especially when it

is used in conjunction with other tests capable of determining cell number

2 One major drawback of the assay is the precipitation of the Neutral Red dye into visible, fine, needle-like crystals When this occurs it is almost impossible to reverse, thus producing inaccurate readings Some chemi- cals induce this precipitation therefore a visual inspection stage in the pro- cedure 1s very important

3 If the intensity of the color is too great, it may be necessary to add a further

2 n-L of resorb solution to some wells If this is required, carry out the procedure for all the wells

3 Riddell, R J., Clothier, R H , and Balls, M (1986) An evaluation of three in vitro cytotoxicity assays Fd Chem Toxic01 24,469-47 1

4 Riddell, R J., Panacer, D S , Wilde, S M., Clothier, R H., and Balls, M (1986) The importance of exposure period and cell type in in vitro cytotoxictty tests ATM

14,86-92

5 Knox, P., Uphill, P F., Fry, J R., Benford, D J., and Balls, M (1986) The FRAME multicentre project on in vitro cytotoxicology Fd Chem Tox 24,457-463

CHAPTER 3

of Cultured Astrocytes for Assay of Gliotoxicity

Mark R Cookson, R McClean, and Vie tor W Pentreatk

1 Introduction Cultured astrocytes provide a valuable and important system for pre- dictive testing and mechanistic analysis of neurotoxic compounds The culture procedures allow relatively rapid assessment of different chemi- cals or their metabolites over a range of concentrations, using cells derived from a restricted source The use of multiwell plates for the cul- tural astrocytes means that multiple samples can be analyzed with a high degree of statistical accuracy and the cell environment can be carefully monitored or manipulated for content of nutrients, ions, agonists, antago- nists, or modulators On the other hand, cultured astrocytes are devoid of their normal integrative functions, and the lack of a blood-brain barrier, the absence of neuron-glial metabolic interactions, and metabolism of substances outside the CNS, together with local regional astrocyte het- erogeneity and limited survival time (about 3 mo) are potential impor- tant shortcomings that require correlative reference to other neuronal, coculture, and in vivo studies A recent review on astrocyte culture for evaluation of neurotoxic-induced injury can be found in ref 1

The preparation and use of astrocytes is a well established and docu- mented procedure for which there is general consensus regarding the principal steps (see refs 2,3) There are, however, many variations in the

From- Methods m Molecular B/ology, Vol 43 In Vitro Toxrcity Testing Protocols

Edited by: S O’Hare and C K Atterwill Copyright Humana Press Inc , Totowa, NJ

17

detailed methodology of, for example, cell separation and media compo- sition, and our descriptions are appropriate for several tests for gliotox- icity Cell purity of the primary cultures are checked by immunostaining for glial fibrillary acidic protein (GFAP), with typical values in the 90- 95% positive range For toxicological studies, subcultured astrocytes are advantageous because higher cell densities and purities may be obtained Keys to the assay and understanding of gliotoxic mechanisms will lie

in the accurate measurement of cell membrane perturbations together with the subsequent intracellular biochemical and metabolic effects Because a large number of physiological and biochemical properties of astrocytes have been measured in culture systems across different disci- plines in neurobiology, a considerable range of potential targets or end- points are available for neurotoxicological evaluation Some examples are described in ref 1 The choice of procedures that may be useful as a preliminary predictive screen is therefore critical and is currently the sub- ject of extensive studies by us To date we have analyzed cell viability, total cell protein, energy utilization, and membrane integrity as likely key indicators These assays can be completed relatively quickly, with cell viability and total protein combined in the same experimental proce- dure and with the energy utilization and membrane integrity also evalu- ated together by uptake and backflux of radiolabeled 2-deoxyglucose (2-DC) The findings show that these have a high degree of validity with, for example, a variety of gliotoxic substances causing increases in total protein (measured in pg/104 cells) at certain critical concentrations How- ever, additional valuable procedures will undoubtedly be described in the future Below we describe convenient methodologies for the prepara- tion of astrocyte cultures and the application of procedures

3 0.1% Trypsin mhibrtor in BME (as above) plus 200 uL deoxyribonuclease

(Type 1, from bovine pancreas) per 5 mL of solution,

4 Serum Supplemented Growth Medium: Dulbecco’s Modified Eagle’s

Medium (DMEM) containing 4500 mg glucose, 110 mg sodium pyruvate, and 110 mg sodium bicarbonate without L-glutamine plus 10% fetal calf

serum and 1% antibiotic solution (see Note 1)

Astrocytes for Gliotoxicity Assay 19

5 Hank’s Balanced Salt Solution with sodium bicarbonate, without calcium chloride or magnesium sulfate (HBSS)

6 TrypsmiEDTA solution: 0.1% made up in HBSS as above

7 Sodium hydroxide: 0.2M

8 Trypan Blue dye solution: 0.4% in 0.8% NaCl and 0.06% K2HP04

9 Coomassie blue based protem assay reagent kit plus bovine serum albumin (BSA) standards (1 mg/mL)

10 2-deoxy-D-[13H] glucose ([3H]2-DG)

11 Ultima Gold High Flashpoint Liquid Scintillation Cocktail

12 Pony vial H/L Miniature polyethylene “Rangin” vials

13 Phosphate buffered saline (PBS), pH 7.4, consisting of in wt/vol; 0.8% NaCl, 0.02% KCl, 0.02% KHZ P04, and 0.115% Na2HP04 made up both with and without 5.6 mM n-glucose (see ref 6)

14 0.2M HCI

3 Methods

1 Poly-L-lysine (PLL) coating: Cells can be grown either on glass cover- slips (13-mm diameter; No I thickness) in 24 well multidishes or in 25 cm2 flasks (see Note 2) Coverslips are flamed after dipping in 95% etha- nol to sterilize, then placed in the wells of the culture dish To each well,

100 pL of poly+lysine solution is added and allowed to dry for 5 min The coverslips are then rinsed with sterile water and allowed to dry thor- oughly before use If coverslips are not required, the PLL can be added directly to the cells of the plate

2 All instruments are sterilized by being flamed in ethanol and are supported

on a piece of aluminum foil in a sterile laminar flow hood Neonatal rat pups are obtained and the heads are quickly wiped in alcohol to sterilize them Pups are sacrificed by decapitation and the heads are placed in a sterile Petri dish with a few milliliters of BME

3 A small cut is made m the skin at the posterior of the skull and the skin removed The skull is very fragile and can be easily removed using two pairs of forceps The whole brain is then removed from the skull cavity by carefully scooping it out

4 The neocortical tissue is isolated by pinching off the olfactory lobes at the anterior and the superior colliculi and the developing cerebellum at the poste- rior of the brain The meninges are teased off and are separated carefully from the cortical hemispheres using fine forceps

5 The cortical tissue is chopped into about eight pieces These are placed in

a sterile tube containing 10 mL of Trypsin/EDTA solution in BME, then are covered and incubated in a water bath at 37°C for 25 min

6 The cells are then triturated using a sterile Pasteur pipet and centrifuged at

9 After discarding the supernatant, 2 mL of supplemented DMEM are added

to resuspend the cells This suspension can be plated out at 50 pL/well or 1 n&/25 cm2 flask

10 After incubating the cells for l-2 h at 37°C in a 5% CO2 humidified atmo- sphere, the cells are fed with 500 pWwel1 or 5 ml/flask of serum supple- mented DMEM These are then incubated for 1 wk and thereafter are fed twice weekly (see Note 3)

11 Staining for Glial Fibrillary Acidic Protein (GFAP) after 1 wk culture (4) should demonstrate that over 90% of such cultures are astrocytes Both anti-GFAP and secondary, fluorescein-conjugated antibodies are commer- cially available However, for various reasons it is often preferable to sub- culture at least once before usmg the cells in toxicological evaluations, mainly to give a greater yield of cultures per rat and to ensure even plating densities across the wells of the multiwell plates

do this depends largely on the density of the cultures and is generally around 5-10 min (see Note 4)

3 Samples from the wells are removed and pooled in a sterile tube, centrr- fuged for 5 min at 1000 rpm, and the supernatant replaced with serum supplemented DMEM as above and plated out at a density of about 2 x lo4 cells/well, as assessed by a hemocytometer count

3.3 Measurement of Cell Viability and Total Protein

1 Cells on coverslips are washed in HBSS three times and are trypsinized wrth 200 /.tL of Trypsm/EDTA m HBSS per well until the cells detach from the coverslips

2 Trypsin inhibitor (150 pLWwel1) IS added and the contents of each removed and placed in labeled microcentrifuge tubes,

Astrocytes for Gliotoxicity Assay 21

3 Of this sample, 50 pL is removed and mixed with 50 l.tL of Trypan blue dye solution, and this sample is counted in a hemocytometer Viability is expressed as percent dye excluding cells divided by the total number of cells

4 The remainder of the cell suspension is centrifuged at 2000 rpm and the supernatant is removed The small pellet of cells is resuspended in 500

pL of 0.2M NaOH, vortexed to disperse the pellet, and left overnight at 4OC

5 BSA standards are made up in the following series; 1, 2.5, 5, 10, 15, 25, and 50 l.tg/mL These are pipeted in duplicate into wells of a microtiter plate (150 pL/well) Likewise, 150 ltL of each sample is added in dupli- cate to a series of wells in the plate, followed by 150 pL of protein assay reagent to each well of the plate (5) The color develops within 5 min at room temperature and lasts for several hours The plates are read at 570 nm using a plate reader (see Note 5)

3.4 Measurement

of Cell Membrane Integrity with PHl2-DG

1 Following the specified incubation with toxicant (see Notes 6 and 7), the medium is aspirated and each well washed twice with 1 mL PBS (37OC)

2 PBS with 5.6 mM o-glucose (450 u,L/well) (37OC) is added followed by 50

PL 0.5 l.tCi/rnL [3H]2-DG Incubation is carried out at 37OC in a 5% CO,/ 95% air humidified incubator for 15-45 min (7)

3 Incubation is terminated by aspiration (with legitimate disposal) of the medium (see Note 8) followed by three washes with 1 mL ice-cold PBS

4 Cells are digested using 300 l.tL 0.2it4 NaOH and left overnight The solu- tion is neutralized with 0.2M HCI

5 Two 150 pL samples are taken directly from the well to the microtiter plate for protein determination and treated as in Section 3.3.5 BSA stan- dards are made up in 0.2M NaCl

6 The remaining solution is transferred to a miniature scintillation vial con- taining 3 mL scintillation cocktail A serial dilution of [3H]2-DG (0.5- 0.0005 l&i) is made up in parallel to act as a standard Samples are shaken and left overnight in the dark at 4OC Samples are counted on a liquid scin- tillation counter (see Note 9)

7 Backflux is determined by incubating cells in PBS with 5 rnM o-glucose containing 0.5 l&i [3H]2-DG as described above At the end of the incuba- tion time the medium is aspirated and each well washed three times with 1

mL PBS (37°C) A further incubation is carried out (15-45 min) in 500 FL PBS without [3H]2-DG This [3H]2-DG solution is treated as in Section 3.4.6 (see Note 10)

8 The remaining cells are treated for protein determination as in Section 3.4., steps 4 and 5 and scmtillation counting as in Section 3.4., step 6

4 Notes

1 Suitable antibiotics are either penicillin/streptomycin (stock 10,000 p, Peni- cillin G, 10 mg streptomycm/mL) or gentamycin (10 mg/mL stock solu- tion) We commonly use the latter

2 Multiwell dishes are convenient for toxicological evaluations since one can perform experiments at five concentrations, plus a control, m quadru- plicate from a 24-well plate

3 It takes around 2 wk for these cells to reach confluency, depending on the age of the rat The fastest growing cultures are prepared from neonates of

~24 h old It is possrble to grow cells from older rats (we have used up to

5 d), but in older rats the more developed meninges are difficult to remove and may contaminate the culture with fibroblast-like cells

4 Time of trypsinization should be kept to a minimum to prevent excess cellular damage The progress of the reaction can be monitored using an inverted microscope

5 Control values for total protein are typically around l-2 l.tg/104 cells

6 We have used 6, 12, and 25 h incubation periods with toxicants Cells are fed within 48 h preceding dosing with toxicant

7 The uptake of [3H]2-DG in older cultures may become reduced We rec- ommend that primary cultures be used soon after confluence (2-3 wk) or 4-5 d after the first subculture

8 Uptake and backflux can be measured together rf Section 3.4., step 7 is proceeded to The total uptake of [3H]2-DG is equal to that contained in the cells plus that in the medium

9 Typical control values for uptake are in the range of l-10 pmol/mg pro- tein/min

10 Typical control values for backflux are between 5 and 10% of uptake

References

1 Aschner, M and Kimelberg, H K (1991) The use of astrocytes in culture as model systems for evaluating neurotoxic-induced-injury Neurotoxicology 12,505-518

2 Shahar, A., de Vellis, J., Vernadakis, A., and Haber, B (eds.) (1989) A Dissection

3 Hertz, L., Juurlink, B H J., Szuchet, S., and Walz, W (1986) In Neuromethods,

Astrocytes for Gliotoxicity Assay 23

5 Redinbaugh, M G and Campbell, W H (1985) Adaptation of the dye binding protein assay to microtitre plates Anal Biochem 147,144-147

6 Brookes N and Yarowsky P J (1985) Determinants of deoxyglucose uptake in cultured astrocytes the role of the sodium pump J Neurochem 44,473-479

7 Yarowsky P J., Boyne A F., Weirwille R., and Brookes N (1986) Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry J Neurosci 6,859-866

CHAPTER 4

Carmel Mothersill

1 Introduction The technique described in this chapter enables the culturing of thy- roid cells without loss of differentiation and medium change It is poten- tially useful for the long-term study of drug effects on the thyroid gland Human thyroid cells obtained during surgery can be maintained in culture for periods of up to 2 mo without losing morphological or func- tional differentiation (1) In the clinical situation the thyroid may be exposed to long-term drug or radiation treatment that may have adverse effects on the functioning of the thyroid gland These adverse effects can

be assessed in culture by studying morphological and biochemical changes after exposure to test chemicals, cytotoxics, or radiation and extrapolated to the likely toxicity in humans

Sections of human thyroid are incubated in a trypsin/collagenase solu- tion The resulting supernatant is filtered and centrifuged twice The cells are collected and resuspended in growth medium and any undigested thyroid tissue is reincubated in the trypsin/collagenase solution on two further occasions, Each supematant is filtered and centrifuged The digests are pooled and plated out in flasks containing Eagle’s Medium The cul- tures are incubated for 48 h and are then exposed to test chemicals and morphology, epithelial cell growth, and biochemical parameters

A long-term culture system for sheep thyroid has been established that retains many of the characteristic functional and morphological features

of the gland The human thyroid culture has been adapted from this sheep culture with only minor modifications Some morphological differentia-

From Methods m Molecular Slotogy, Vol 43 In Vltfo Toxmty Testing Protocols

Edlted by S O’Hare and C K Atterw~ll Copyright Humana Press Inc., Totowa, NJ

25

tion time discrepancies occur Follicles develop in sheep cultures in 5-8

d and in human cultures after 15-20 d Undifferentiated areas are more common in human cultures and are visualized as patches of epithelial cells devoid of follicles

The unusual glucose and lactate metabolism of the sheep system per- mits a prolonged culture period Glucose is rapidly metabolized to lac- tate, and then the lactate is utilized by the cultures over their remaining life-span Exhaustion of lactate in the culture medium coincides with cell death, but the latter can be delayed by adding concentrated glucose to the medium just before this occurs The metabolism of glucose to lactate and subsequent lactate utilization, follows the same pattern in human cultures, but at a much slower rate because of the lesser degree of differentiation (lactate use correlates strongly with morphological differentiation) The major factor in establishing a human thyroid culture is the amount and character of healthy tissue obtained The best cultures are from 5-10 g samples of thyroid tissue, although even samples of 0.05 g have been cultured The slower rate of differentiation of the human culture system

is advantageous when long-term studies of drugs or radiation effects on the human thyroid are required The test chemical can be added directly above the differentiated monolayer without disturbing the media or the degree of differentiation that occurs Although human thyroid cultures have been established and utilized by other scientists, they have been short-term systems or subcultures maintained by the use of hormones or growth enhancers In general, these have been used to study the bio- chemical behavior of the cultures in the short-term or in the characteris- tics of the subculture In these cases the primary culture was not maintained for more than 7 d

This culture system correlates well with the in vivo situation Thyroid cultures have a limited life-span in humans (15-20 doublings), which equates to the deterioration of cells in culture after the third or fourth subculture The best endpoints for determining that the cells are func- tioning correctly are the T4 assay or 1251 trapping ability

The thyroid culture shows a progressive loss of differentiation after repeated subculture It is postulated that this may be a result of the effects

of the trypsination, which causes the release of a receptor component into the medium that binds thyrotrophin This receptor is regenerated when the cells are replated, but it is thought that the regeneration declines after repeated subcultures

Human Thyroid Culture 27

A large number of scientists use whole animals or animal cell culture systems (mostly rodent) that have limited use in relation to the study of human disease and toxicity The heterogeneity of the source material in terms of genetic makeup and previous history of cytotoxic insult is a disadvantage in relation to the development of a standardized routine test system

2 Materials

1 Normal and diseased human thyroid tissue excised during surgery

2 Blood administrator set

3 100 j.t.m fine surgical gauze

4 Centrifuge

5 40 mL sealed flasks

6 Sterile scalpel

7 Radioimmunoassay kit for T4

8 Liquid scintillation counter

18 Growth medium: 500 mL Eagle’s medium, 2 mM L-glutamine, 20% v/v lamb serum, 0.1 ug/mL hydrocortisone, 10 mL U/n-& insulin, 1 @4 potas- sium iodide, 20 IU/mL penicillin, 20 pg/mL streptomycin, 4 IU/mL gentamicin, 1 pg/rnL fungizone, 40 mL U/r& thyrotrophin, 12.5 mL 1M HEPES buffer

3 Methods 3.1 Culture Procedure

1 Add pieces of human thyroid to ice-cold BSS continumg antibiotrcs Chop the thyroid into small pieces, preferably of 5-10 g, using a sterile scalpel

Add the chopped tissue to 12 mL trypsin/collagenase solution at 37°C Incubate for 30 min at 37OC

2 Filter the supernatant (containing freed cells) through a blood admmistra- tion set Filter again through a fine surgical gauze, thus removing any fat and fibrous debris Centrifuge the filtrate at 4OOg

3 Resuspend the tissue digest in an equal volume of growth medium con- taining 40% serum, neutralizing trypsin activity

4 Incubate any undigested thyroid pieces m the trypsin/collagenase solution and incubate for two further periods of incubation (depending on the size

of the tissue) followed by filtration, Centrifuge

5 Pool the cells from all three digests Count the cells and adjust a 0.5 mL cell suspension to -1 x lo6 cells Add the adjusted cell suspension to 5 mL

of growth medium in a 40 mL flask

6 Plate directly into a flask containing the minimum amount of medium necessary to wet the surface of the plastic Leave for 24 h (the explant should have adhered) Add further medium up to a total volume of 5 mL

3.2 Testing The following assays can be performed to evaluate any thyrotoxicity

1 After 48 h expose the cultures to test chemicals (0.05-0.1 mL in culture medium or DMSO/ethanol), cytotoxics, or irradiation as follows:

a 5 replicates for each chemical

d Use a T4 radioimmunoassay kit to monitor T4 release into the medium

e Iodine trapping ability: Add 0.1 mL of 2 mM methimazole and 0.1

mL of 2 pCi/mL sodium 125I to the medium of the cell culture at room temperature, Incubate for 90 min Wash the culture thoroughly with

4 aliquots of 5 mL Earle’s BSS to remove all traces of free radioactivity Trypsinize the cells with 5 mL trypsin/EDTA solution and resuspend in -10 mL fresh growth medium Count the cells and determine the 1251 levels

by liquid scintillation Calculate the 1251 counts/106 cells Plot a graph of

1251 counts/lO”cells as a function of time in culture for the differentiated cultures

Human Thyroid Culture 29

1

DAYS IN CULTURE Fig 1 The levels of T, (ng/106 cells) detected in medium from differentiated huan thyroid cultures (dertved from multinodular goiter) over a 40-d life span (n = 6)

3 Epithelial cell outgrowth: Monitor the tissue outgrowth by measuring its area and by performing autoradiography Study the effects of the toxic agents on the different cell types present using immunocytochemical analy- sis for intermediate filaments or surface antigens

3.3 Results

3.3.1 T4 Plot a standard curve for T4 Read off the values for T4 found in the medium samples at regular time intervals Plot a graph of T4 levels over

40 d (Fig 1)

3.3.2 Iodine Trapping Ability Calculate the 125I counts/106 cells Plot a graph of 1251 counts/106 cells

as a function of time in culture for the differentiated culture (Fig 2)

4 Notes

1 If the tissue sample is small, incubate with the trypsin/collagenase solution and then plate directly mto a flask After 48 h, expose the cultures to test chemicals as above

2 Fibroblast contamination can occur in cultures derived from low initial cell numbers Seeding high numbers of cells probably inhibits the prolif- eration of any fibroblasts present

3 If the amount of tissue is very small (co.5 cm3), incubate the tissue sample with trypsin/collagenase solutron for 30 mm at 37°C

4 Cultures rapidly metabolize the available glucose after which cell death occurs, therefore, it may be necessary to prolong the culture life-span by adding 0.1 mL of a concentrated glucose solution This can be assessed initially by using a glucose assay, or once experienced with the technique,

by judging the color of the medium

Human Thyroid Culture 31

of recent experimental thyroidology A major aim of such endeavors has been to facilitate the development of simple, reliable, reproducible test- ing strategies for compounds interacting with, and perturbing the func- tion of, the thyroid follicular cell (TFC) The earliest experimental thyroid models were based on organ culture or tissue slice preparations

or, alternatively, short-term cell suspensions Subsequently, however, it has become possible to maintain TFCs as monolayer cultures in which a high level of thyroid-specific differentiation and responsiveness may be preserved over a prolonged period Such characteristics, which allow experimental intervention and the subsequent study of cell function and morphology, have facilitated the development of biological assays for thyrotrophin (TSH) (I) and thyroid autoantibodies (2) in serum, and have recently begun to encompass applications in the field of cellular toxicol- ogy, where the application of these new investigative tools has enabled the identification of the sites and mechanisms of action of agents demon- strating direct thyroid toxicity in vivo (3)

The preservation of TSH-dependent responses in cultured TFCs, together with the relative ease with which large numbers of identical,

From Methods in Molecular Biology, Vol 43: In Vitro Toxicity Testing Protocols

Edited by S O’Hare and C K Atterwill Copynght Humana Press Inc., Totowa, NJ

33

replicate cultures may be maintained, has made the TFC monolayer the model of choice for the quantitative assessment of the agents interfering with or modifying TSH-receptor interaction, transmembrane iodide movement, or cell proliferation The most widely adopted in vitro func- tional markers of TFC stimulation have included intracellular CAMP accumulation and thyroid-radioiodide uptake, the latter being a response unique to the thyroid cell

Although cell proliferation within the normal adult thyroid gland is minimal, growth is enhanced by dietary goitrogens, such as the cyono- genie glucosides and thioglucosides, which inhibit iodiode uptake, lead- ing to an impaired synthesis of thyroid hormones and a compensatory rise in pituitary TSH secretion In vitro strategies for estimating the rate

of cell proliferation involve the determination of the incorporation of C3H] thymidine into subconfluent TFC monolayers or, alternatively, assessment of the metaphase index of the culture (i.e., determination of the percentage of cells with chromosomes visibly in the “S” phase) (4) Despite the widespread use of primary thyroid cultures derived directly from thyroid tissue as a fundamental tool in thyroid cell biology, the inher- ent viability in agonist and antagonist responsiveness between prepara- tions of cells derived from different individual thyroid tissues, coupled with the progressive dedifferentiation of cultures with increasing dura- tion of in vitro maintenance, has limited the use of this system to essen- tially short-term, qualitative investigations of thyroid function and proliferation, Recently, however, a number of stable cell lines have been isolated that retain major features of the differentiated follicular cell Foremost among these is FRTL-5, a cloned Fischer rat thyroid cell (5-7)

2 Materials

2.1 Basic Maintenance Medium

FRTL-5 cell monolayers are maintained in Ham’s F- 12 medium (Coon’s modification) containing various supplements, as described below Each liter

of working medium is prepared by adding 2.5 g NaHC03 to 12.08 g of powdered medium and making this to 1 L with triple glass-distilled water The medium is then filtered (0.22 pm) into presterilized 100 mL bottles and may be stored at 4°C for up to 3 mo Immediately prior to use, a sup- plement of 4 hormones (as detailed below), MEM nonessential amino acids, penicillin and streptomycin are filtered into the medium

Thyroid Toxicity Testing In Vitro 35

2.2 Hormone Supplements The hormones comprising this supplement are somatostatin (1 mg/L), hydrocortisone (0.33 mg/L), transferrin (OS g/L), and glycyl-histidyl- lysine acetate (2 mg/L) The stock supplement is prepared as follows:

1 Somatostatin: 50 ug is dissolved in 500 uL of Ca2+h4g2+ free Hank’s bal- anced salt solution (HBSS), and then made to 5 mL with Ca2+/Mg2+ free HBSS

2 Hydrocortisone: 1 mg is dissolved into 1.5 rnL absolute ethanol, and 100

uL then is added to 10.9 mL Ca2+/Mg2+ free HBSS

3 Glycyl-histidyl lycine acetate: 1 mg is dissolved in 1 mL Ca2+/Mg2+ free HBSS

4 Transferrin: 25 mg is dissolved into 5 mL Ca2+/Mg2+ free HBSS

To prepare stock aliquots of the combined hormone supplement, 5 rnL

of each of the solutions detailed above is added to 25 rnL Ca2+h4g2+ free HBSS, mixed and stored as 1 mL aliquots at -70°C until required After thawing, each is diluted in 100 mL of basic maintenance medium The preparation of complete maintenance medium also requires addition of

200 mmol glutamine/L, nonessential amino acids (stored as 1 mL aliquots at 4”C), penicillin (100 U/mL) and streptomycin (100 pg/niL) (stored as 1 mL aliquots at -2OOC) Immediately before use, supplements are filtered (0.22 pm pore size) into 90 rnL of base medium, and sterile newborn calf serum (NCS) added

2.3 TSH Preparations

A number of biologically active preparations of TSH are available from commercial sources, which may be used as reference thyroid cell stimulators Bovine TSH (First International Standard of Thyrotrophin) (pituitary TSH, bovine, for bioassay; coded 53/11) and human TSH (Second International Standard, coded 80/558) are specifically recom- mended for this purpose, and may be obtained from the National Insti- tute for Biological Standards and Control (South Mimms, Potters Bar, Herts, UK) Both are supplied as ampuled, lyophilized preparations having a uniform, stated activity, and are stable at -20°C over prolonged periods (i.e., years) After reconstitution, preparations are stored as ali- quots at -7O”C, and used within 6 mo It is particularly important that partially used aliquots are not refrozen, since this will diminish their biological activity

Fig 1 Phase-contrast photomicrograph of a monolayer colony of the rat thy- roid follicular cell strain FRTL-5,48 h after passaging (200x magnification)

2.4 FRTL-5 Cells FRTL-5 cells are available from the American Type Culture Collec- tion (Rockville, MD) They are routinely passaged in Coon’s modified Ham’s F12 medium supplemented with the “4H” hormone mixture described above, together with 10 pg/mL insulin, 100 pU/mL TSH, and 5% (v/v) NCS Cells are grown in lo-cm diameter Petri dishes in 5% COz in air at 37°C In the presence of TSH, the cells proliferate as uni- form, round colonies (Fig 1)

3.1 Preparation of Cell Suspension

from Stock FRTL-5 Cultures

1 Aseptically aspirate the growth medium from stock cultures of FRTL-5 cells Rinse the cultures with prewarmed Ca2+/Mg2+-free HBSS

Thyroid Toxicity Testing In Vitro 37

2 To each culture, add a sterile solution of trypsm (1 mg/mL) and collage- nase (20 U/n& in Ca*+/Mg*+-free HBSS) Ensure that the liquid covers the monolayer, Return the cultures to the incubator for 2-5 min

3 Use a sterile Pasteur pipet to transfer the suspension of detached cells into

a sterile 25 mL universal tube Clumped cells should be dispersed by repeatedly pipetmg the suspension

4 Add calf serum (0.5% v/v) to inactivate the trypsin

5 Close the tube and shake gently to obtain a uniform cell suspension

6 Centrifuge (lOOg, 5 min at room temperature) to obtain a cell pellet

7 Remove the supernatant solution with a sterile Pasteur pipet, and resus- pend the cells to an appropriate density in a small volume (e.g., 5 mL) of the plating medium

3.2 Preparation of Replicate FRTL-5 Cell Monolayers

1, Having prepared a suspension of single, viable FWI’L-5 cells from stock cul- tures, initiate the replicate monolayers that will form the bioassay “target” tissue by adding aliquots of the cell suspension to 24-well tissue culture dishes

2 After initiating the test cultures maintain the bioassay plates at 37°C under 5% CO2 in air, in a water-saturated atmosphere to prevent evaporation of the culture medium

3.3 Prebioassay Treatment of Cells

1 A change of culture medium will be necessary 3-4 d after initiation of cultures Remove the exhausted medium from the monolayers using a ster- ile Pasteur pipet attached to a vacuum suction pump and collection jar

2 Add fresh medium (500 pLWwel1) to each monolayer with a minimum of delay, so that the cultures do not become dry

3 Imtiate the bioassay by the addition of test solutions at the time of, or shortly after the first medium change

3.4 Procedure for Extracting

Assessment of the effect of a compound on TSH-dependent adenylate cyclase activity is made by determining the final intracellular CAMP lev- els attained in the presence of a serial dilution of that compound, in cells simultaneously exposed to a standard dose of TSH, compared with the CAMP level attained in cells exposed to TSH alone

1 Remove the maintenance medium from subconfluent monolayers by asep- tic aspiration, and replace with 500 pL Leibovitz (L-15) medium

2 Add 3-isobutyl-1-methylxanthine (IBMX) to a final level of 0.4 mM m all wells, to inhibit CAMP-dependent phosphodiesterase activity

diluent to triplicate sets of incubation wells

4 Add a standard dose of TSH (e.g., 100 pU/mL) to each well

5 After incubation for 15 min, remove the medium, and add 500 ltL of ice- cold absolute ethanol to each culture This treatment both arrests the incu- bation reaction and releases intracellular CAMP from the lysed cells

6 Seal the culture plates in wrapping film to prevent evaporation of ethanol, and transfer them to a -20°C freezer for 24 h

7 Remove 200 p,L aliquots of the ethanolic fractions and transfer these to small glass test tubes

8 Evaporate the tube contents to dryness under a stream of nitrogen

9 Redissolve the dried residues in 25 mM Tris, 50 mM NaCl, 8 n&f or other appropriate assay buffer

10 Determine the CAMP content by conventional radioimmunoassay (e.g., usmg a commercial kit)

11, Express the final CAMP level attained within each set of triplicate cultures

6 After incubation, remove the radioactive media and carefully discard Briefly rinse (X2) each culture with 50 pL ice-cold HBSS

7 After washing, add 500 l.tL 100 pJ4 sodium perchlorate to each well This inhibits iodide pump activity, allowing intracellular iodide to discharge into the medium

8 After 20 min, remove duplicate 100 l.tL portions of the sodium perchlorate solution, and determine 1251 content using a y-scintillation counter Express

Thyroid Toxicity Testing In Vitro

results as a percentage of the mean 1251 uptake value obtained in replicate cultures exposed to the standard TSH dose alone

3.6 PH] Thymidine Incorporation

as a Marker of Cell Proliferation

1 Remove the maintenance medium from subconfluent monolayers by asep- tic aspiration, and replace with a fresh 500 pL aliquot of the same medium

4 Terminate the incubation by removal of the medium

5 Briefly rinse each culture (X2) with ice-cold 10% (w/v) trichloroacetic acid (TCA), followed by a further addition of 500 pL 10% TCA/well Leave the cultures for 3-4 h at 4°C to allow protein precipitation

6 Remove the acid supernatants using a fresh Pasteur pipet for each well Add 250 pL of 1N NaOH/L to each well Seal the plates, wrap in aluminum foil, and leave overnight at room temperature to allow cellular digestion

7 Remove duplicate 100 pL aliquots, and transfer these to scintillation vials Add liquid scintillant (e.g., “Hisafe 2” scintillant, Pharmacia, Uppsala, Sweden) (4 mL) to each vial and determine [3H] thymidine activity using a p-scintillation counter

3.7 Metaphase Index Determination

1 Follow the procedure shown in 3.5 for iodide uptake to include step 4

2 After incubation for 44 h, remove the medium and add 200 PL fresh, serum-free maintenance medium together with 30 pL colcemid, a mitotic- spindle arresting agent

3 Incubate cultures for a further 3 h at 37°C

4 Remove the incubation medium, and add 200 pL of freshly-prepared 30% (w/v) glacial acetic acid/70% ethanol to fix the cells

5 After 15 min, remove the fixative with a Pasteur pipet, and rinse the mono- layers twice in 70% ethanol Leave to dry overnight

6 After drying, add 100 pL diluted Giemsa stain to each well Leave for 150

mm, replenishing the stain after 1 h

7 Rmse the cell layers twice in 70% ethanol, then once with 95% ethanol

8 Dry the cell layers, and observe for metaphase figures under a 40x objective

9 Calculate the Metaphase Index (X/100) x lOO%, where X= no of cells/100 displaying figures

4 Notes

1 Since the uniformity of the monolayer test cultures is crucial in obtaming a high level of precision and sensitivity of the assay system, cells must be dispensed into the culture wells with the aid of a fixed-volume repeating pipet, using disposable micropipet tips previously sterilized by autoclav- ing or y-irradiation Assuming an even distrtbution of smgle cells within the starting suspension, tt 1s possible to obtain a between-culture variation

in cell plating density approaching & l-2% In the case of standard 24-well plates, a suitable starting inoculum may consist of lo5 cells/well

2 L-15 medium does not require an equilibrating CO2 gas phase, so that incubations may be performed in room air at 37°C

3 It is important to recogmze that the calculation in Section 3.4., step 11, assumes that the population densities of cells within replicate cultures are closely identical (i.e., within Z!Z l-2% limit) The between-culture variation in density may be estimated on the basis of cell protem or DNA estimations in a separate series of cultures wtthin each batch of assay plates

4 One of the major advantages of the FRTL-5 is that unlike primary cultures, these cells may be maintained in long-term culture, having a reproducible, fully characterized behavioral pattern and population-doubling time Given stable culture conditions, therefore, the responses of sequential passages

of FRTL-5 cells should be entirely predictable This has the important advantage of enabling large numbers of replicate and uniformly respon- sive test monolayers to be established, while also generating the inocula of FRTL-5 cells required to initiate subsequent stock cultures

5 It may be desirable to investigate, m parallel cultures, for actions of TFC function on both CAMP accumulatton and transmembrane iodide uptake Thus, although the latter is dependent on the functtonal and structural integrity of the cell membrane, specific inhibition of iodide pump or Na+/ K+-dependent ATPase activity may not necessarily, at least in the short term, adversely affect adenylate cyclase activity in a cell membrane that is otherwise structurally intact However, if both CAMP accumulation and iodide uptake are diminished after incubation of cells with the test mate- rial, an effect on cell viability should be suspected

6 In order to investigate for thyroid-directed toxic actions of nonwater soluble molecules, after solubilizing these in a nonpolar solvent, it is impor- tant that equivalent levels of the solvent are also included within TSH- containing control cultures