Mutation Research, 31 (1975) I35-148 @) Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands
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HUMAN P E R I P H E R A L BLOOD LYMPHOCYTES FOR T H E ANALYSIS OF CHROMOSOME ABERRATIONS IN MUTAGEN TESTS
H. J. EVANS AND M A U R E E N L. O'RIORDAN
Medical Research Council, Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh (Great Britain) (Received December I2th, 1974)
I. SOME ADVANTAGES AND DISADVANTAGES
Studies on exposed individuals, and on cultured cells, have shown that the human peripheral blood lymphocyte is an extremely sensitive indicator of both in viva and in vitro induced chromosome structural change. These changes in chromosome structure offer readily scored morphological evidence of damage to the genetic material. Although problems exist in the extrapolation from in vitro results to the in vivo situation, the lymphocyte offers several advantages as a test system: (I) Easy availability of large numbers of human cells: a few ml of peripheral blood can be easily and repeatedly obtained from an individual and each I ml of blood can contain 1-3" lO 6 small lymphocytes. (2) The lymphocytes are distributed throughout the body, circulate in all tissues and a proportion are long-lived. (3) Virtually all the peripheral blood lymphocytes are a synchronised cell population in the same Go or Glstage of mitotic interphase, and, in healthy individuals, these cells are only infrequently involved in mitotic proliferation in vivo. (4) A proportion of the lymphocytes can be stimulated by mitogens to undergo mitosis in culture; they are easy to culture and thus provide a ready source of dividing cells for the scoring of chromosome aberrations. (5) Thele are excellent techniques available for making chromosome preparations from lymphocytes and the cells have a low spontaneous chromosome aberration frequency. Some disadvantages include: (I) Although the lymphocytes are a synchronised population, there are different sub-populations of cells even within the same individual. These can have varying responses to different mitogenic agents and probably differing intermitotic times which may be further influenced by variations in culture conditions. (2) As in other cell systems, there is the possibility of non-uniformity of exposure and of preferential growth in culture of less heavily damaged cells. (3) It is now recognised that in man only T-lymphocytes, i.e. the thymicderived cells, can be stimulated in culture by phytohaemagglutinin to undergo mitosis. The T-lymphocyte is closely concerned with the immune response, thus, where an analysis of an in vivo exposure is to be undertaken, any recent previous exposure
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of an individual to an immunological stimulus may positively or negatively alter the number of cells with chromosome aberrations, depending on when the cells are withdrawn from the individual. These disadvantages relate largely to the use of lymphocytes for studying mutagen effects in vivo and are of less relevance to the use of lymphocytes for in vitro mutagen testing. II. TYPES OF DAMAGE ASSAYED
The types of chromosome damage which can be cytologically distinguished at metaphase can be divided into two main groups: chromosome-type and chromatidtype. The circulating lymphocyte is in the Go or G1 phase of mitosis and exposure to ionising radiations and certain other mutagenic agents during this stage produces chromosome-type damage where the unit of breakage and reunion is the whole chromosome (i.e. both chromatids at the same locus). Howevec, cells exposed to these agents while in the S oc G2 stages of the cell cycle, after the chromosome has divided into two sister chromatids, yield chromatid-type aberrations and only the single chromatid is involved in breakage or exchange (Fig. I). Other agents (e.g. some of the alkylating agents) will produce only chromatid-type aberrations although the cells are exposed to the mutagen whilst in Go or G1.
(A ) Chromosome-type aberrations Studies on somatic metaphases show that seven classes of chromosome-type aberrations can be distinguished cytologically (Fig. 2). Aberration types 1- 5 involve only a single chromosome and are known as intrachanges whereas types 6 and 7 involve the exchange of parts between chromosomes and therefore are classified as
interchanges. (z) Terminal deletions (Fig. 3). These are paired acentric fragments which have the appearance of resulting from a simple break across the chromosome and they are not associated with an obvious exchange aberration. (2) Minutes (interstitial, isodiametric or dot deletions) (Fig. 4)- These are pairs of acentric fragments, smaller in size than terminal deletions, characteristically appearing as paired spheres of chromatin. These are intercalary deletions. (3) Acentric rings (Fig. 5). These are paired segments of chromatid without a centromere and which are joined to give a ring. (4) Centric rings (Fig. 6). Ring structures containing a centromere. The centric ring can be easily distinguished morphologically from the acentric type and is generally accompanied by one acentric fragment. (5) Inversions (Fig. 7). Chromosome inversions can be classified into two categories: (a) paracentric inversions where both points of breakage and reunion lie on the same arm of the chromosome; and (b) pericentric inversions where the points of breakage and inversion lie on opposite sides of the centromere. In cells where the two points of pericentric exchange are of unequal distance from the centromere, the abnormal chromosome can be easily distinguished by the altered position of the centromere. However, where the exchange points are equally distant from the centromere, and in the case of paracentric inversions, there is no change in the relative position of the centromere. The detection of such inversions
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CHROMOSOME-TYPE X
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CPIROMATID-TYPE
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Fig. i. Relation between type of aberration and stage in cell cycle at the time of exposure to irradiation or certain mutagens. NORMAL
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CENTRIC TERMINAL INTERSTITIAL RING AND DELETION DELETION FRAGMENT
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ACENTRIC PERICENTRIC RING INVERSION
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DIGENTRIC AND FRAGMENT
SYMMETRICAL INTERCHANGE
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Fig. 2. Seven classes of chromosome-type aberrations cytologically distinguishable at mitotic metaphase.
Fig. 3. Cell with a terminal deletion. Fig. 4. Cell with a minute intercalary deletion. m a y , therefore, be possible only t h r o u g h t h e use of chromosome b a n d i n g techniques. (6) Reciprocal translocations ( s y m m e t r i c a l interchange) (Fig. 8). These are a b e r r a t i o n s which involve b r e a k a g e of two chromosomes a n d t h e reciprocal exchange of b r o k e n s e g m e n t s between these chromosomes. Conventional staining m e t h o d s will n o t allow t h e d e t e c t i o n of reciprocal t r a n s l o c a t i o n s involving t h e exchange of two equal-sized segments, a n d b a n d e d c h r o m o s o m e p r e p a r a t i o n s are required. I n t e r changes m a y , of course, also occur in the centromere regions of two chromosomes giving w h o l e - a r m exchanges which, if t h e y involve acrocentric chromosomes, are s o m e t i m e s referred to as centric fusions ( R o b e r t s o n i a n translocations).
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Fig. 5- Cell with acentric rings. Fig. 6. Cell with centric ring and fragment.
Fig. 7- Cell with a pericentric inversion in a C group chromosome. Fig. 8. Cell with reciprocal translocation.
Fig. 9. ceil with dicentric c h r o m o s o m e and acentric fragment.
(7) Dicentric or polycentric aberrations ( a s y m m e t r i c a l interchange) (Fig. 9). A b e r r a t i o n s which arise from an exchange b e t w e e n two or more chromosomes which results in t h e centric p r o d u c t s r e u n i t i n g in such a w a y to form a dicentric or polycentric s t r u c t u r e a n d an a s s o c i a t e d acentric fragment. A b e r r a t i o n t y p e s ~i), (2) a n d (3) i.e. t e r m i n a l deletions, m i n u t e s a n d acentric rings, are often loosely g r o u p e d t o g e t h e r as acentric f r a g m e n t s which are n o t associated with a n y obvious exchange event resulting in a r e a r r a n g e m e n t . F r a g m e n t s associated with an exchange event e.g. the f r a g m e n t observed in association with a
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dicentric, are scored as part of the exchange and not as separate aberrations in their own right.
(B) Chromatid-type aberrations Chromatid-type aberrations are induced by ionising radiations when cells are exposed in the S or G2 stages of interphase, chromosome-type damage being produced in cells irradiated in G1. However, m a n y chemical agents and viruses cause only chromatid-type damage even though the cells are exposed to the agent whilst ~n the G1 phase and examined in their first subsequent division. These aberrations are a consequence of errors in replication that occur at the DNA synthesis, or S, phase following exposure. In contrast to the chromosome-type aberrations, chromatid breaks and particularly gaps are unreliable indicators of real damage to the genetic material. Not only can the scoring of gaps and breaks be extremely subjective, resulting in considerable observer differences, but also m a n y gaps are caused by technical artefacts e.g. "poor" culture conditions, and the use of "drastic" processes during slide preparation. For this reason, if chromatid gaps and breaks are to be scored and included in the aberration yield, it is particularly important that "control" blood cultures are cultured in a similar way and at the same time as those exposed to the agent being tested. (I) Chromatid and isochromatid gap (Fig. IO). The gap or achromatic lesion appears as a non-staining and constricted region in the chromatid arm and the apparently "broken" segments of the chromatid area are in alignment. Where the gap involves both chromatid arms at the same position, this is referred to as an isolocus or isochromatid gap. (2) Chromatid break (Fig. ii). Where there is a discontinuity with displacement in the chromatid arm so that the broken chromatid ends are not aligned. An apparently "simple" break results in a terminal deletion. (3) Chromatid minutes. Single, or unpaired, intercalary fragments. (4) Chromatid acentric rings. Intercalary fragments joined to give ring structures. (5) Centric rings. Intrachanges resulting in chromatid ring structures in which the centromere is included in the ring. (6) Inversions. Paracentric inversions are not readily scorable, but pericentric inversions m a y be detected because of the close pairing of chromatids.
Fig. IO. Cell with chromatid gap. Fig. I I. Cell with chromatid break.
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aberration result is the time the cells are maintained in culture. After two days in culture at 37 ° the majority of transformed lymphocytes are undergoing their first mitosis. Preparations made from cells cultured for longer periods will contain increasing proportions of cells in their second or subsequent divisions. Consequently, if lymphocytes with chromosome-type damage fail to undergo repeated division in vitro, due to their genetic unbalance or mechanical difficulties at anaphase, examination of cells after 72 h of culture may not reflect the true in vivo aberration level. Furthermore, a proportion of the chromosome-type aberrations observed at this later time may be really of a "derived" type resulting from a duplication of aberrations that were initially chromatid-type (Fig. 14). Thus, for tests which are designed to detect the presence or absence of chromosome damage in lymphocytes following in vivo exposure of individuals to a suspected mutagenic agent, the blood samples should only be cultured for up to 50 h at 37 °. The same restriction applies to in vitro studies, but consideration must also be given to the fact that the agent and the test may itself inhibit cell development and so prolong the time of culture necessary to obtain mitotic cells. III. A P P A R A T U S N E E D E D
Small autoclave for sterilisation. Temperature controlled water bath or incubator in which temperature can be controlled to ~ I °. Bench centrifuge. Microscope; good quality light microscope capable of magnifying 15oo times. Refrigerator, preferably with deep-freeze. Still or deionising equipment. Filter sterilising system. Sterile glass or plastic syringes in sizes between 1-2o ml. Disposable needles to fit syringes with bore sizes between 19 gauge and 2I gauge. Sterile glass or plastic containers with screw caps: (a) for collection of blood; (b) for the blood cultures; (c) for sterile liquids. Glass or plastic containers for non-sterile liquids. Centrifuge tubes with polythene or rubber stoppers. Glass pipettes. Glass microscope slides and coverslips. Staining dishes and slide racks. IV. C H E M I C A L M A T E R I A L S R E Q U I R E D
Anticoagulant: Lithium heparin. Mitogen : Phytohaemagglutinin. Spindle inhibitor: Demecolcine (colcemid) 0.02% solution, or Colchicine or Vinblastine (velban). Tissue culture media: Virtually any standard tissue culture medium, e.g. Hams FIo; McCoy's; TC 199; Eagle's Minimum Essential Medium (MEM). Serum: A wide variety of sera can be used. Human A B + or bovine (calf, but not necessarily foetal calf) are amongst the most popular.
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Antibiotics: Penicillin IOO I U / m l of medium; Streptomycin IOO I U / m l of medium. p H control: Sodium bicarbonate; 5% COs in air. Hypotonic solutions: Potassium chloride 0.075 M solution; Sodium citrate 0.95 % solution ; Hanks' solution: diluted I : 4 with distilled water. Fixatives: Methanol; Glacial acetic acid. Stains: Lactic aceto-orcein, 1% or 2% solution in I : I lactic acid (70% aqueous)glacial acetic acid; Giemsa 2% (aqueous solution). Cleaning agents: Cellosolve; Xylene ; Euparal essence. Mountants: Euparol; De Pe X. For "in vitro" tests: Known chemical mutagen as a positive control, e.g. nitrogen mustard or triethylenemelamine. V. COSTING
A very approximate figure for the cost of a single test culture and its analysis can be estimated. Each test will involve a number of cultures at different dose levels, positive and negative controls and replicates. Other expenses which should be considered include the expenditure on basic equipment (III), accommodation and electricity costs. In addition it is not easy to assess the cost involved in obtaining blood samples. An estimated cost for one culture is given based on using IO ml of culture medium and the analysis of IOO cells. Reagents Culture medium, mitotic inhibitor, hypotonic solution, fixative, stain: Estim a t e d cost $ L5O Equipment Disposable syringes and needles, disposable containers, microscope slides and coverslips, pipettes: Estimated cost $ I.oo Time Culture handling, slide preparation: Analvsis of cells:
o.5h 4.0 h 4.5 man hours
VI. PREPARATION OF CULTURE MEDIUM
It is preferable to prepare the culture medium in advance and then store at 4 ° until required. Each new batch of medium should be tested for sterility and compared with previous batches for ability to support cell growth. In practice it is often easier to prepare sufficient medium in bulk for a whole experiment rather than prepare small individual aliquots. This also helps to minimise technical artefacts which can arise from variation in different batches of medium. A suitable starting quantity of culture medium, i.e. sufficient for 45 cultures and using IO ml medium for each culture, consists of: 4oo ml proprietary tissue culture medium.
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5.0 1111phytohaemagglutinin. 7° ml serum. optional - streptomycin, IOO IU/ml of medium. Penicillin, IOO IU/ml of medium. A variety of tissue culture medium can be used with success (see IV) but, in our hands, Ham's FIo has been found to yield the highest and Eagle's MEM the lowest, number of dividing cells in a 48-h culture. The pH of the final culture medium should be between 6.8 and 7.2; sodium bicarbonate or 5% COs in air may be used to adjust the pH. After preparation, Io-ml or 5-ml aliquots of cultuce medium should be dispensed into sterile glass or plastic screw-capped vials. Where the larger quantity is used, a 3o-ml container is recomlp.ended, and a I5-ml vial is suitable for the 5-ml aliquots. The choice between the two largely depends on the number of cells which are finally required, but the Io-ml amount generally yields a superior final result. -
VII. C O L L E C T I O N O F B L O O D S A M P L E S
(a) Choice o/ subject for study The relationship between the frequency of chromosome aberrations detected in an individual's cultured lymphocytes and the level of his in vivo exposure to the chemical being specifically tested, may be complicated by other environmental factors: for example, previous or current exposure to prescribed and unprescribed drugs, diagnostic or therapeutic radiation exposure, viral infections. It is therefore particularly important to collect adequate data for each individual being examined on: age, occupational history, radiation and drug histories, exposure to toxic substances, e.g. organic solvents, insecticides. For in vivo studies, a similarly sized control population is required to provide background data on the frequency of chromosome aberrations. Ideally, control individuals should have a closely similar environmental background and be sex- and age-matched with the "exposed" persons. Any batch of blood samples should contain a mixture of samples from both control and exposed groups and these should be processed together. In this way any differences between groups that may be due to, or influenced by, technical artefacts, e.g. variations in culture medium, fluctuations in incubation temperature and cell fixation, will be minimised. Where persons are being selected to provide blood samples for an in vitro test, medical data should also be collected, but it is preferable to exclude individuals suffering from virus or other infections or who have received excessive doses of radiation or drugs, or recently received immunisation. Wherever possible, it is also advisable to use blood samples from several individuals to minimise the possibility of a varying response between the lymphocytes of different individuals and the chemical being tested. (b) Method Blood samples (5-1o ml) for both in vivo and in vitro test systems are normally taken from adults by venipuncture using a lO-2O ml sterile syringe. The blood is then immediately transferred to a suitable sized sterile glass, or plastic, vessel containing lithium heparin (preservative-free) at concentrations of between IO-IOO IU
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of heparin/i ml of blood. The specimen is then gently mixed to prevent clotting. If the blood is not immediately required for culture, it should be stored at between 4-25 °. It is advisable to set up the cultures within 24 h of withdrawal, since delay m a y adversely affect cell viability. To prevent observer bias in the in vivo tests, the blood samples from the control and the exposed persons should be randomised and coded by someone who is not concerned in the study. Coding of samples in the in vitro test system should be done after slide preparation. It is mcst important that the scorer has no prior knowledge of which slides are from exposed and which from control cultures, so that all slides are coded and then randomised before being presented to the scorer. VIII. PE RIPHERAL BLOOD LYMPHOCYTE CULTURE TECHNIQUE
Two methods of culturing the peripheral blood lymphocytes can be used.
(a) Macroculture method This is not a recommended technique since it requires relatively large quantities of blood. After gently centrifuging the blood sample, the plasma is separated off from the red cells and cultured as below. (b) Microculture method (i) The heparinised blood sample, unless freshly withdrawn, should be gently shaken to allow proper mixing of the cells and plasma. (2) 0.8 ml of whole blood is added to io nil of culture medium in a 3o-ml sterile glass or plastic vial, which should be then tightly closed by a screw cap. Where 5 ml of medium is used, only 0. 4 ml of blood should be used for each culture. (02) The culture is then incubated at 37 ° in a water bath or incubator for 455 ° h. The cultures are not shaken during incubation. (4) During the last 3 h prior to harvesting the cells, a spindle inhibitor should be added to the culture. Since spindle inhibitors can be toxic to cells in high concentration, it is advisable to ascertain in advance the most suitable concentration. Many laboratories use demecolcine to give a final concentration in the culture of 0.2/~g/ml, or colchicine to give 0.25 #g/ml. IX. HARVESTING THE CULTURE
(I) Time of harvesting, see II(C). (2) The cells and culture medium should be removed from the incubator, gently mixed and then the contents emptied into a centrifuge tube. (3) The cells are spun down b y gentle centrifugation for 5 min at approx. 2ooo-25oo rev./min (approx. 6o ×g). The cells should be then in a loose button at the b o t t o m of the tube and the supernatant clear. (4) The supernatant is gently poured off and the button of cells shaken and resuspended in approx, io ml of the hypotonic solution (we routinely use the o.o75 M potassium chloride hypotonic solution). The suspension should then be allowed to stand at either room temperature or at 37 ° for 8 to IO min. (5) The culture should be centrifuged as before and the supernatant decanted
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off. The cells are resuspended and 5 ml of fixative (3:1 methanol-glacial acetic acid) should be slowly added using a small pipette, whilst agitating the cells. (6) After centrifugation two further changes of fixative are recommended and the cells should be exposed to fixative at least 15 rain before the slides are made. The required cells can be satisfactorily stored in the second or third change of fixative for several days before slides are made, although this is not recommended. If cells are stored in fixative a further change of fresh fixative will be necessary immediately prior to slide preparation. The fixative should always be freshly made up and not stored for periods of time longer than one hour. X. S L I D E P R E P A R A T I O N
The cells are suspended in fresh fixative to give a suitable, slightly cloudy suspension. One or two drops of suspension should be dropped on to a clean greasefree microscope slide and left to dry. The ambient temperature and humidity affects the degree of cell spreading. A relative humidity of 45-65% is found to give best results. It m a y be necessary to speed up the air-drying process by vigorously waving the slide through a spirit flame. The fixative on no account should be allowed to ignite, otherwise the cells tend to break. The slides should then be stained with aceto-orcein, or Giemsa, cleared and finally mounted. xI. I n vitro TESTS (a) General aspects The major problem in the in vitro testing of suspected mutagenic agents is the assessment of the result and the extrapolation of either the positive or negative cytogenetic findings to the in vivo situation. It is well known that a number of chemicals which are apparently inactive mutagens in vitro have a cytogenetically positive effect in vivo, due to their metabolic activation. Conversely, other compounds produce a positive in vitro effect even at small dosage levels, but have a reduced in vivo activity. I n vitro tests can therefore only provide a limited guide to the possible in vivo effects of a suspected mutagen. (b) Method (z) Blood samples and cultures. If possible, blood samples from several individuals should be used for in vitro testing. This ensures that any variability in results due to possible inherent differences between individuals is taken into account. Two kinds of control are recommended and these should be cultured in parallel, using the same batches of medium and incubation times, to ensure the adequacy of the system and to exclude the possibilities of false negatives or the presence of unknown contaminants. For the "positive" control, a known chromosome breaking agent, e.g. triethylenemelamine should be added to the cultures at various concentrations. "Negative" controls are provided by cultures to which "carrier" (saline) but no mutagen, has been added. Three dose levels of the agent under test are recommended: (i) the m a x i m u m
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tolerated concentration which allows the cells to undergo mitosis, (if) a concentration comparable to the expected in vivo physiological level, (iii) an intermediate concentration. (2) Exposure and harvesting time. Many chemical agents produce "delayed" effects so that aberrations are only evident in the second or subsequent mitosis after the cells have been exposed to the mutagen. In addition, the different stages of the cell cycle m a y show large variations in sensitivity to the induction of chromosome damage, and some agents cause mitotic delay. For example a good positive control involves exposure of cultures to nitrogen mustard (Mustine hydrochloride), at a final concentration of 3' lO-6 M in the culture, for 30 rain at 37 °, 24 to 36 h after culture initiation. Such an exposure, however, results in a delay in cell development, so that the maxim u m yields of first division cells are observed in cultures incubated for 65 to 75 h and in 72-h cultures some 40 to 60% of the cells contain chromatid structural changes. (3) Chromosome aberration analysis. In good preparations, generally those with a high mitotic index, the ceils selected for analysis tend to be of a superior quality than those from poorer preparations. To avoid bias in cell selection, certain criteria should be defined and adhered to inespective of the overall quality of the preparations. The following suggestions are given as guide lines: (a) The coded slides should be methodically scanned. (b) With the low power objective, a metaphase spread which appears to be in an unbroken cell should be selected for analysis. (c) Under high power, the chromosomes should be well defined and should not be in an early C-anaphase state with completely separated chromatids. (d) To avoid the analysis oi cells with random chromosome loss due to technical artefacts, only cells with a minimum number of 45 centromeres should be scored. (e) If the cell is suitable for analysis the vernier reading should be recorded to locate the cell for possible future reference. (f) The chromosomes should be then counted and any aberrations noted. Where difficulty is found in the interpretation of an aberration, the cell should not be rejected but the difficulty recorded and the opinion oi a second observe,- sought. The cells can be fully counted and analysed in detail (karyotyped) but this is an extremely time-consuming procedure and m a y be not necessary to obtain a valid estimate of aberration yield. Alternatively, the chromosomes in each cell should be counted and the presence of any chromosome- or chromatid-type damage recorded. All unstable types ot chromosome aberrations, e.g. dicentrics, rings and fragments, are particularly good parameters of damage since they are all easily identifiable. It may not be profitable to spend time attempting to analyse reciprocal translocations, since they are more difficult to detect. It is more profitable and informative to spend that time scoring more cells for the readily visible fiagments and asymmetrical exchanges. A recommended minimum number of IOO cells (see X I I ) should be scored from each of the duplicate blood culture treatments. A record should be kept of all cell references, whether or not aberrations were found. XII. STATISTICAL T R E A T M E N T
The chromosome aberrations induced by m a n y mutagens m a y have a random
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distribution between cells, although they may be non-randomly distributed w i t h i n chromosomes. Consequently the number of cells with o, I, 2. . . . . . n aberrations may conform with a Poisson distribution, where the mean and its variance are approximately equal. The mean frequency of aberrations may be small and the smaller the mean the larger will be the number of cells to be scored to obtain good estimates of aberration yield for meaningful comparisons with both negative and positive controls. However, since slides are scored blind, observers usually aim to score a fixed number of cells per slide e.g. ioo metaphases with say two slides per culture (or 5 ° metaphases on each of four slides). Replication is important to estimate "between slides", "between culture" and "between treatment" variances. A simple guide for determining the approximate numbers of cells to be scored (or aimed for) at anticipated, or known, aberration frequency levels in order to detect a significant result, may be obtained by simply calculating (or looking up in tables) binomial confidence limits. For example: if P is the proportion of abnormal cells (cells with aberrations) and Q = I - - P then the approximate 95% confidence limits of P are given by:
P ± tA/PQ where t is the upper 2.5% point on the normal curve and n is the number of cells to be scored. For 95% limits t -- 1.96; for 80% limits t = 1.28 etc. e.g. for a frequency of 0.20 (20% of cells abnormal) after scoring IOO cells, the 95% confidence limits of that mean estimate are: 0.2 :]: 1.96(o.o4), or 0.2 :~ 0.08 = 0.28 to o.12 If 400 cells are scored, and P -- 0.20, the approximate limits (95%) are 0.24 to o.16. Standard significance tests are employed to test the null hypothesis of a difference between results from negative control and treated cultures. In this way the conclusion that a given substance is, or is not, mutagenic in the test described can therefore be made in a quantitative fashion with defined levels of probability. In some cases, for instance in studying the effects of different dose levels of a known mutagen, such as ionizing radiations, it may be required to express the relationship between dose (D) and aberration yield (y), where y will be some function of D, in the form of the parameters of an equation representing the dose-response curve. For example this could be a linear function where y = a+blD
where a is the spontaneous aberration yield in control cultures and b is a constant. Alternatively, the response may be defined by a quadratic: y = a+blD+b~D ~
where bl and b~ are constants. Or by a power law: y = a+blD ~
here, assuming a to be negligible, the logarithm of the aberration frequency is linearly related to the logarithm of the dose, i.e. log y = log b 1 + n log D.
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T h e c o n s t a n t s for t h e s e e q u a t i o n s for a n y p a r t i c u l a r s e t of d o s e - r e s p o n s e d a t a are calculated using least squares regression methods and various computer prog r a m m e s a r e a v a i l a b l e f o r a n u m b e r of d o s e - r e s p o n s e m o d e l s . XIII. LITERATURE BUCKTON, K. E., AND I-t. J. EVANS (Eds.), Methods for the Analysis of Human Chromosome Aberrations, World Health Organisation, Geneva, 1973. COHEN, M. M., AND I~. HIRSCHHORN, Cytogenetic studies in animals, in Chemical iUIutagens. Principles and Methods for their Detection, A. HOLLAENDER (Ed.), VO1. 2, Plenum, New York, 1971 . DE SERRES, F. J., AND W. SHERIDAN (Eds.), The evaluation of chemical mutagenicity data in relation to population risk, in Environmental Health Perspectives, Exp. Issue No. 6, US Department of Health, Education and Welfare, Washington D.C., 1973. EVANS, I-i. J., Chromosome aberrations induced by ionizing radiation, Int. Rev. Cytol., 13 (1962) 22I 23I.
EVANS, H. J., Population cytogenetics and environmental factors, in Pfizer Medical 3/Ionographs 5, Edinburgh University Press, 197 ° , pp. 192-216. KIHLMAN, B. A., Actions of Chemicals on Dividing Cells, Prentice Hall, Englewood Cliffs, N.J., 1966.
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HUMAN P E R I P H E R A L BLOOD LYMPHOCYTES FOR T H E ANALYSIS OF CHROMOSOME ABERRATIONS IN MUTAGEN TESTS
H. J. EVANS AND M A U R E E N L. O'RIORDAN
Medical Research Council, Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh (Great Britain) (Received December I2th, 1974)
I. SOME ADVANTAGES AND DISADVANTAGES
Studies on exposed individuals, and on cultured cells, have shown that the human peripheral blood lymphocyte is an extremely sensitive indicator of both in viva and in vitro induced chromosome structural change. These changes in chromosome structure offer readily scored morphological evidence of damage to the genetic material. Although problems exist in the extrapolation from in vitro results to the in vivo situation, the lymphocyte offers several advantages as a test system: (I) Easy availability of large numbers of human cells: a few ml of peripheral blood can be easily and repeatedly obtained from an individual and each I ml of blood can contain 1-3" lO 6 small lymphocytes. (2) The lymphocytes are distributed throughout the body, circulate in all tissues and a proportion are long-lived. (3) Virtually all the peripheral blood lymphocytes are a synchronised cell population in the same Go or Glstage of mitotic interphase, and, in healthy individuals, these cells are only infrequently involved in mitotic proliferation in vivo. (4) A proportion of the lymphocytes can be stimulated by mitogens to undergo mitosis in culture; they are easy to culture and thus provide a ready source of dividing cells for the scoring of chromosome aberrations. (5) Thele are excellent techniques available for making chromosome preparations from lymphocytes and the cells have a low spontaneous chromosome aberration frequency. Some disadvantages include: (I) Although the lymphocytes are a synchronised population, there are different sub-populations of cells even within the same individual. These can have varying responses to different mitogenic agents and probably differing intermitotic times which may be further influenced by variations in culture conditions. (2) As in other cell systems, there is the possibility of non-uniformity of exposure and of preferential growth in culture of less heavily damaged cells. (3) It is now recognised that in man only T-lymphocytes, i.e. the thymicderived cells, can be stimulated in culture by phytohaemagglutinin to undergo mitosis. The T-lymphocyte is closely concerned with the immune response, thus, where an analysis of an in vivo exposure is to be undertaken, any recent previous exposure
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of an individual to an immunological stimulus may positively or negatively alter the number of cells with chromosome aberrations, depending on when the cells are withdrawn from the individual. These disadvantages relate largely to the use of lymphocytes for studying mutagen effects in vivo and are of less relevance to the use of lymphocytes for in vitro mutagen testing. II. TYPES OF DAMAGE ASSAYED
The types of chromosome damage which can be cytologically distinguished at metaphase can be divided into two main groups: chromosome-type and chromatidtype. The circulating lymphocyte is in the Go or G1 phase of mitosis and exposure to ionising radiations and certain other mutagenic agents during this stage produces chromosome-type damage where the unit of breakage and reunion is the whole chromosome (i.e. both chromatids at the same locus). Howevec, cells exposed to these agents while in the S oc G2 stages of the cell cycle, after the chromosome has divided into two sister chromatids, yield chromatid-type aberrations and only the single chromatid is involved in breakage or exchange (Fig. I). Other agents (e.g. some of the alkylating agents) will produce only chromatid-type aberrations although the cells are exposed to the mutagen whilst in Go or G1.
(A ) Chromosome-type aberrations Studies on somatic metaphases show that seven classes of chromosome-type aberrations can be distinguished cytologically (Fig. 2). Aberration types 1- 5 involve only a single chromosome and are known as intrachanges whereas types 6 and 7 involve the exchange of parts between chromosomes and therefore are classified as
interchanges. (z) Terminal deletions (Fig. 3). These are paired acentric fragments which have the appearance of resulting from a simple break across the chromosome and they are not associated with an obvious exchange aberration. (2) Minutes (interstitial, isodiametric or dot deletions) (Fig. 4)- These are pairs of acentric fragments, smaller in size than terminal deletions, characteristically appearing as paired spheres of chromatin. These are intercalary deletions. (3) Acentric rings (Fig. 5). These are paired segments of chromatid without a centromere and which are joined to give a ring. (4) Centric rings (Fig. 6). Ring structures containing a centromere. The centric ring can be easily distinguished morphologically from the acentric type and is generally accompanied by one acentric fragment. (5) Inversions (Fig. 7). Chromosome inversions can be classified into two categories: (a) paracentric inversions where both points of breakage and reunion lie on the same arm of the chromosome; and (b) pericentric inversions where the points of breakage and inversion lie on opposite sides of the centromere. In cells where the two points of pericentric exchange are of unequal distance from the centromere, the abnormal chromosome can be easily distinguished by the altered position of the centromere. However, where the exchange points are equally distant from the centromere, and in the case of paracentric inversions, there is no change in the relative position of the centromere. The detection of such inversions
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CHROMOSOME-TYPE X
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137
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Fig. i. Relation between type of aberration and stage in cell cycle at the time of exposure to irradiation or certain mutagens. NORMAL
ou
CENTRIC TERMINAL INTERSTITIAL RING AND DELETION DELETION FRAGMENT
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ACENTRIC PERICENTRIC RING INVERSION
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SYMMETRICAL INTERCHANGE
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Fig. 2. Seven classes of chromosome-type aberrations cytologically distinguishable at mitotic metaphase.
Fig. 3. Cell with a terminal deletion. Fig. 4. Cell with a minute intercalary deletion. m a y , therefore, be possible only t h r o u g h t h e use of chromosome b a n d i n g techniques. (6) Reciprocal translocations ( s y m m e t r i c a l interchange) (Fig. 8). These are a b e r r a t i o n s which involve b r e a k a g e of two chromosomes a n d t h e reciprocal exchange of b r o k e n s e g m e n t s between these chromosomes. Conventional staining m e t h o d s will n o t allow t h e d e t e c t i o n of reciprocal t r a n s l o c a t i o n s involving t h e exchange of two equal-sized segments, a n d b a n d e d c h r o m o s o m e p r e p a r a t i o n s are required. I n t e r changes m a y , of course, also occur in the centromere regions of two chromosomes giving w h o l e - a r m exchanges which, if t h e y involve acrocentric chromosomes, are s o m e t i m e s referred to as centric fusions ( R o b e r t s o n i a n translocations).
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Fig. 5- Cell with acentric rings. Fig. 6. Cell with centric ring and fragment.
Fig. 7- Cell with a pericentric inversion in a C group chromosome. Fig. 8. Cell with reciprocal translocation.
Fig. 9. ceil with dicentric c h r o m o s o m e and acentric fragment.
(7) Dicentric or polycentric aberrations ( a s y m m e t r i c a l interchange) (Fig. 9). A b e r r a t i o n s which arise from an exchange b e t w e e n two or more chromosomes which results in t h e centric p r o d u c t s r e u n i t i n g in such a w a y to form a dicentric or polycentric s t r u c t u r e a n d an a s s o c i a t e d acentric fragment. A b e r r a t i o n t y p e s ~i), (2) a n d (3) i.e. t e r m i n a l deletions, m i n u t e s a n d acentric rings, are often loosely g r o u p e d t o g e t h e r as acentric f r a g m e n t s which are n o t associated with a n y obvious exchange event resulting in a r e a r r a n g e m e n t . F r a g m e n t s associated with an exchange event e.g. the f r a g m e n t observed in association with a
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dicentric, are scored as part of the exchange and not as separate aberrations in their own right.
(B) Chromatid-type aberrations Chromatid-type aberrations are induced by ionising radiations when cells are exposed in the S or G2 stages of interphase, chromosome-type damage being produced in cells irradiated in G1. However, m a n y chemical agents and viruses cause only chromatid-type damage even though the cells are exposed to the agent whilst ~n the G1 phase and examined in their first subsequent division. These aberrations are a consequence of errors in replication that occur at the DNA synthesis, or S, phase following exposure. In contrast to the chromosome-type aberrations, chromatid breaks and particularly gaps are unreliable indicators of real damage to the genetic material. Not only can the scoring of gaps and breaks be extremely subjective, resulting in considerable observer differences, but also m a n y gaps are caused by technical artefacts e.g. "poor" culture conditions, and the use of "drastic" processes during slide preparation. For this reason, if chromatid gaps and breaks are to be scored and included in the aberration yield, it is particularly important that "control" blood cultures are cultured in a similar way and at the same time as those exposed to the agent being tested. (I) Chromatid and isochromatid gap (Fig. IO). The gap or achromatic lesion appears as a non-staining and constricted region in the chromatid arm and the apparently "broken" segments of the chromatid area are in alignment. Where the gap involves both chromatid arms at the same position, this is referred to as an isolocus or isochromatid gap. (2) Chromatid break (Fig. ii). Where there is a discontinuity with displacement in the chromatid arm so that the broken chromatid ends are not aligned. An apparently "simple" break results in a terminal deletion. (3) Chromatid minutes. Single, or unpaired, intercalary fragments. (4) Chromatid acentric rings. Intercalary fragments joined to give ring structures. (5) Centric rings. Intrachanges resulting in chromatid ring structures in which the centromere is included in the ring. (6) Inversions. Paracentric inversions are not readily scorable, but pericentric inversions m a y be detected because of the close pairing of chromatids.
Fig. IO. Cell with chromatid gap. Fig. I I. Cell with chromatid break.
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aberration result is the time the cells are maintained in culture. After two days in culture at 37 ° the majority of transformed lymphocytes are undergoing their first mitosis. Preparations made from cells cultured for longer periods will contain increasing proportions of cells in their second or subsequent divisions. Consequently, if lymphocytes with chromosome-type damage fail to undergo repeated division in vitro, due to their genetic unbalance or mechanical difficulties at anaphase, examination of cells after 72 h of culture may not reflect the true in vivo aberration level. Furthermore, a proportion of the chromosome-type aberrations observed at this later time may be really of a "derived" type resulting from a duplication of aberrations that were initially chromatid-type (Fig. 14). Thus, for tests which are designed to detect the presence or absence of chromosome damage in lymphocytes following in vivo exposure of individuals to a suspected mutagenic agent, the blood samples should only be cultured for up to 50 h at 37 °. The same restriction applies to in vitro studies, but consideration must also be given to the fact that the agent and the test may itself inhibit cell development and so prolong the time of culture necessary to obtain mitotic cells. III. A P P A R A T U S N E E D E D
Small autoclave for sterilisation. Temperature controlled water bath or incubator in which temperature can be controlled to ~ I °. Bench centrifuge. Microscope; good quality light microscope capable of magnifying 15oo times. Refrigerator, preferably with deep-freeze. Still or deionising equipment. Filter sterilising system. Sterile glass or plastic syringes in sizes between 1-2o ml. Disposable needles to fit syringes with bore sizes between 19 gauge and 2I gauge. Sterile glass or plastic containers with screw caps: (a) for collection of blood; (b) for the blood cultures; (c) for sterile liquids. Glass or plastic containers for non-sterile liquids. Centrifuge tubes with polythene or rubber stoppers. Glass pipettes. Glass microscope slides and coverslips. Staining dishes and slide racks. IV. C H E M I C A L M A T E R I A L S R E Q U I R E D
Anticoagulant: Lithium heparin. Mitogen : Phytohaemagglutinin. Spindle inhibitor: Demecolcine (colcemid) 0.02% solution, or Colchicine or Vinblastine (velban). Tissue culture media: Virtually any standard tissue culture medium, e.g. Hams FIo; McCoy's; TC 199; Eagle's Minimum Essential Medium (MEM). Serum: A wide variety of sera can be used. Human A B + or bovine (calf, but not necessarily foetal calf) are amongst the most popular.
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Antibiotics: Penicillin IOO I U / m l of medium; Streptomycin IOO I U / m l of medium. p H control: Sodium bicarbonate; 5% COs in air. Hypotonic solutions: Potassium chloride 0.075 M solution; Sodium citrate 0.95 % solution ; Hanks' solution: diluted I : 4 with distilled water. Fixatives: Methanol; Glacial acetic acid. Stains: Lactic aceto-orcein, 1% or 2% solution in I : I lactic acid (70% aqueous)glacial acetic acid; Giemsa 2% (aqueous solution). Cleaning agents: Cellosolve; Xylene ; Euparal essence. Mountants: Euparol; De Pe X. For "in vitro" tests: Known chemical mutagen as a positive control, e.g. nitrogen mustard or triethylenemelamine. V. COSTING
A very approximate figure for the cost of a single test culture and its analysis can be estimated. Each test will involve a number of cultures at different dose levels, positive and negative controls and replicates. Other expenses which should be considered include the expenditure on basic equipment (III), accommodation and electricity costs. In addition it is not easy to assess the cost involved in obtaining blood samples. An estimated cost for one culture is given based on using IO ml of culture medium and the analysis of IOO cells. Reagents Culture medium, mitotic inhibitor, hypotonic solution, fixative, stain: Estim a t e d cost $ L5O Equipment Disposable syringes and needles, disposable containers, microscope slides and coverslips, pipettes: Estimated cost $ I.oo Time Culture handling, slide preparation: Analvsis of cells:
o.5h 4.0 h 4.5 man hours
VI. PREPARATION OF CULTURE MEDIUM
It is preferable to prepare the culture medium in advance and then store at 4 ° until required. Each new batch of medium should be tested for sterility and compared with previous batches for ability to support cell growth. In practice it is often easier to prepare sufficient medium in bulk for a whole experiment rather than prepare small individual aliquots. This also helps to minimise technical artefacts which can arise from variation in different batches of medium. A suitable starting quantity of culture medium, i.e. sufficient for 45 cultures and using IO ml medium for each culture, consists of: 4oo ml proprietary tissue culture medium.
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5.0 1111phytohaemagglutinin. 7° ml serum. optional - streptomycin, IOO IU/ml of medium. Penicillin, IOO IU/ml of medium. A variety of tissue culture medium can be used with success (see IV) but, in our hands, Ham's FIo has been found to yield the highest and Eagle's MEM the lowest, number of dividing cells in a 48-h culture. The pH of the final culture medium should be between 6.8 and 7.2; sodium bicarbonate or 5% COs in air may be used to adjust the pH. After preparation, Io-ml or 5-ml aliquots of cultuce medium should be dispensed into sterile glass or plastic screw-capped vials. Where the larger quantity is used, a 3o-ml container is recomlp.ended, and a I5-ml vial is suitable for the 5-ml aliquots. The choice between the two largely depends on the number of cells which are finally required, but the Io-ml amount generally yields a superior final result. -
VII. C O L L E C T I O N O F B L O O D S A M P L E S
(a) Choice o/ subject for study The relationship between the frequency of chromosome aberrations detected in an individual's cultured lymphocytes and the level of his in vivo exposure to the chemical being specifically tested, may be complicated by other environmental factors: for example, previous or current exposure to prescribed and unprescribed drugs, diagnostic or therapeutic radiation exposure, viral infections. It is therefore particularly important to collect adequate data for each individual being examined on: age, occupational history, radiation and drug histories, exposure to toxic substances, e.g. organic solvents, insecticides. For in vivo studies, a similarly sized control population is required to provide background data on the frequency of chromosome aberrations. Ideally, control individuals should have a closely similar environmental background and be sex- and age-matched with the "exposed" persons. Any batch of blood samples should contain a mixture of samples from both control and exposed groups and these should be processed together. In this way any differences between groups that may be due to, or influenced by, technical artefacts, e.g. variations in culture medium, fluctuations in incubation temperature and cell fixation, will be minimised. Where persons are being selected to provide blood samples for an in vitro test, medical data should also be collected, but it is preferable to exclude individuals suffering from virus or other infections or who have received excessive doses of radiation or drugs, or recently received immunisation. Wherever possible, it is also advisable to use blood samples from several individuals to minimise the possibility of a varying response between the lymphocytes of different individuals and the chemical being tested. (b) Method Blood samples (5-1o ml) for both in vivo and in vitro test systems are normally taken from adults by venipuncture using a lO-2O ml sterile syringe. The blood is then immediately transferred to a suitable sized sterile glass, or plastic, vessel containing lithium heparin (preservative-free) at concentrations of between IO-IOO IU
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of heparin/i ml of blood. The specimen is then gently mixed to prevent clotting. If the blood is not immediately required for culture, it should be stored at between 4-25 °. It is advisable to set up the cultures within 24 h of withdrawal, since delay m a y adversely affect cell viability. To prevent observer bias in the in vivo tests, the blood samples from the control and the exposed persons should be randomised and coded by someone who is not concerned in the study. Coding of samples in the in vitro test system should be done after slide preparation. It is mcst important that the scorer has no prior knowledge of which slides are from exposed and which from control cultures, so that all slides are coded and then randomised before being presented to the scorer. VIII. PE RIPHERAL BLOOD LYMPHOCYTE CULTURE TECHNIQUE
Two methods of culturing the peripheral blood lymphocytes can be used.
(a) Macroculture method This is not a recommended technique since it requires relatively large quantities of blood. After gently centrifuging the blood sample, the plasma is separated off from the red cells and cultured as below. (b) Microculture method (i) The heparinised blood sample, unless freshly withdrawn, should be gently shaken to allow proper mixing of the cells and plasma. (2) 0.8 ml of whole blood is added to io nil of culture medium in a 3o-ml sterile glass or plastic vial, which should be then tightly closed by a screw cap. Where 5 ml of medium is used, only 0. 4 ml of blood should be used for each culture. (02) The culture is then incubated at 37 ° in a water bath or incubator for 455 ° h. The cultures are not shaken during incubation. (4) During the last 3 h prior to harvesting the cells, a spindle inhibitor should be added to the culture. Since spindle inhibitors can be toxic to cells in high concentration, it is advisable to ascertain in advance the most suitable concentration. Many laboratories use demecolcine to give a final concentration in the culture of 0.2/~g/ml, or colchicine to give 0.25 #g/ml. IX. HARVESTING THE CULTURE
(I) Time of harvesting, see II(C). (2) The cells and culture medium should be removed from the incubator, gently mixed and then the contents emptied into a centrifuge tube. (3) The cells are spun down b y gentle centrifugation for 5 min at approx. 2ooo-25oo rev./min (approx. 6o ×g). The cells should be then in a loose button at the b o t t o m of the tube and the supernatant clear. (4) The supernatant is gently poured off and the button of cells shaken and resuspended in approx, io ml of the hypotonic solution (we routinely use the o.o75 M potassium chloride hypotonic solution). The suspension should then be allowed to stand at either room temperature or at 37 ° for 8 to IO min. (5) The culture should be centrifuged as before and the supernatant decanted
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off. The cells are resuspended and 5 ml of fixative (3:1 methanol-glacial acetic acid) should be slowly added using a small pipette, whilst agitating the cells. (6) After centrifugation two further changes of fixative are recommended and the cells should be exposed to fixative at least 15 rain before the slides are made. The required cells can be satisfactorily stored in the second or third change of fixative for several days before slides are made, although this is not recommended. If cells are stored in fixative a further change of fresh fixative will be necessary immediately prior to slide preparation. The fixative should always be freshly made up and not stored for periods of time longer than one hour. X. S L I D E P R E P A R A T I O N
The cells are suspended in fresh fixative to give a suitable, slightly cloudy suspension. One or two drops of suspension should be dropped on to a clean greasefree microscope slide and left to dry. The ambient temperature and humidity affects the degree of cell spreading. A relative humidity of 45-65% is found to give best results. It m a y be necessary to speed up the air-drying process by vigorously waving the slide through a spirit flame. The fixative on no account should be allowed to ignite, otherwise the cells tend to break. The slides should then be stained with aceto-orcein, or Giemsa, cleared and finally mounted. xI. I n vitro TESTS (a) General aspects The major problem in the in vitro testing of suspected mutagenic agents is the assessment of the result and the extrapolation of either the positive or negative cytogenetic findings to the in vivo situation. It is well known that a number of chemicals which are apparently inactive mutagens in vitro have a cytogenetically positive effect in vivo, due to their metabolic activation. Conversely, other compounds produce a positive in vitro effect even at small dosage levels, but have a reduced in vivo activity. I n vitro tests can therefore only provide a limited guide to the possible in vivo effects of a suspected mutagen. (b) Method (z) Blood samples and cultures. If possible, blood samples from several individuals should be used for in vitro testing. This ensures that any variability in results due to possible inherent differences between individuals is taken into account. Two kinds of control are recommended and these should be cultured in parallel, using the same batches of medium and incubation times, to ensure the adequacy of the system and to exclude the possibilities of false negatives or the presence of unknown contaminants. For the "positive" control, a known chromosome breaking agent, e.g. triethylenemelamine should be added to the cultures at various concentrations. "Negative" controls are provided by cultures to which "carrier" (saline) but no mutagen, has been added. Three dose levels of the agent under test are recommended: (i) the m a x i m u m
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tolerated concentration which allows the cells to undergo mitosis, (if) a concentration comparable to the expected in vivo physiological level, (iii) an intermediate concentration. (2) Exposure and harvesting time. Many chemical agents produce "delayed" effects so that aberrations are only evident in the second or subsequent mitosis after the cells have been exposed to the mutagen. In addition, the different stages of the cell cycle m a y show large variations in sensitivity to the induction of chromosome damage, and some agents cause mitotic delay. For example a good positive control involves exposure of cultures to nitrogen mustard (Mustine hydrochloride), at a final concentration of 3' lO-6 M in the culture, for 30 rain at 37 °, 24 to 36 h after culture initiation. Such an exposure, however, results in a delay in cell development, so that the maxim u m yields of first division cells are observed in cultures incubated for 65 to 75 h and in 72-h cultures some 40 to 60% of the cells contain chromatid structural changes. (3) Chromosome aberration analysis. In good preparations, generally those with a high mitotic index, the ceils selected for analysis tend to be of a superior quality than those from poorer preparations. To avoid bias in cell selection, certain criteria should be defined and adhered to inespective of the overall quality of the preparations. The following suggestions are given as guide lines: (a) The coded slides should be methodically scanned. (b) With the low power objective, a metaphase spread which appears to be in an unbroken cell should be selected for analysis. (c) Under high power, the chromosomes should be well defined and should not be in an early C-anaphase state with completely separated chromatids. (d) To avoid the analysis oi cells with random chromosome loss due to technical artefacts, only cells with a minimum number of 45 centromeres should be scored. (e) If the cell is suitable for analysis the vernier reading should be recorded to locate the cell for possible future reference. (f) The chromosomes should be then counted and any aberrations noted. Where difficulty is found in the interpretation of an aberration, the cell should not be rejected but the difficulty recorded and the opinion oi a second observe,- sought. The cells can be fully counted and analysed in detail (karyotyped) but this is an extremely time-consuming procedure and m a y be not necessary to obtain a valid estimate of aberration yield. Alternatively, the chromosomes in each cell should be counted and the presence of any chromosome- or chromatid-type damage recorded. All unstable types ot chromosome aberrations, e.g. dicentrics, rings and fragments, are particularly good parameters of damage since they are all easily identifiable. It may not be profitable to spend time attempting to analyse reciprocal translocations, since they are more difficult to detect. It is more profitable and informative to spend that time scoring more cells for the readily visible fiagments and asymmetrical exchanges. A recommended minimum number of IOO cells (see X I I ) should be scored from each of the duplicate blood culture treatments. A record should be kept of all cell references, whether or not aberrations were found. XII. STATISTICAL T R E A T M E N T
The chromosome aberrations induced by m a n y mutagens m a y have a random
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distribution between cells, although they may be non-randomly distributed w i t h i n chromosomes. Consequently the number of cells with o, I, 2. . . . . . n aberrations may conform with a Poisson distribution, where the mean and its variance are approximately equal. The mean frequency of aberrations may be small and the smaller the mean the larger will be the number of cells to be scored to obtain good estimates of aberration yield for meaningful comparisons with both negative and positive controls. However, since slides are scored blind, observers usually aim to score a fixed number of cells per slide e.g. ioo metaphases with say two slides per culture (or 5 ° metaphases on each of four slides). Replication is important to estimate "between slides", "between culture" and "between treatment" variances. A simple guide for determining the approximate numbers of cells to be scored (or aimed for) at anticipated, or known, aberration frequency levels in order to detect a significant result, may be obtained by simply calculating (or looking up in tables) binomial confidence limits. For example: if P is the proportion of abnormal cells (cells with aberrations) and Q = I - - P then the approximate 95% confidence limits of P are given by:
P ± tA/PQ where t is the upper 2.5% point on the normal curve and n is the number of cells to be scored. For 95% limits t -- 1.96; for 80% limits t = 1.28 etc. e.g. for a frequency of 0.20 (20% of cells abnormal) after scoring IOO cells, the 95% confidence limits of that mean estimate are: 0.2 :]: 1.96(o.o4), or 0.2 :~ 0.08 = 0.28 to o.12 If 400 cells are scored, and P -- 0.20, the approximate limits (95%) are 0.24 to o.16. Standard significance tests are employed to test the null hypothesis of a difference between results from negative control and treated cultures. In this way the conclusion that a given substance is, or is not, mutagenic in the test described can therefore be made in a quantitative fashion with defined levels of probability. In some cases, for instance in studying the effects of different dose levels of a known mutagen, such as ionizing radiations, it may be required to express the relationship between dose (D) and aberration yield (y), where y will be some function of D, in the form of the parameters of an equation representing the dose-response curve. For example this could be a linear function where y = a+blD
where a is the spontaneous aberration yield in control cultures and b is a constant. Alternatively, the response may be defined by a quadratic: y = a+blD+b~D ~
where bl and b~ are constants. Or by a power law: y = a+blD ~
here, assuming a to be negligible, the logarithm of the aberration frequency is linearly related to the logarithm of the dose, i.e. log y = log b 1 + n log D.
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T h e c o n s t a n t s for t h e s e e q u a t i o n s for a n y p a r t i c u l a r s e t of d o s e - r e s p o n s e d a t a are calculated using least squares regression methods and various computer prog r a m m e s a r e a v a i l a b l e f o r a n u m b e r of d o s e - r e s p o n s e m o d e l s . XIII. LITERATURE BUCKTON, K. E., AND I-t. J. EVANS (Eds.), Methods for the Analysis of Human Chromosome Aberrations, World Health Organisation, Geneva, 1973. COHEN, M. M., AND I~. HIRSCHHORN, Cytogenetic studies in animals, in Chemical iUIutagens. Principles and Methods for their Detection, A. HOLLAENDER (Ed.), VO1. 2, Plenum, New York, 1971 . DE SERRES, F. J., AND W. SHERIDAN (Eds.), The evaluation of chemical mutagenicity data in relation to population risk, in Environmental Health Perspectives, Exp. Issue No. 6, US Department of Health, Education and Welfare, Washington D.C., 1973. EVANS, I-i. J., Chromosome aberrations induced by ionizing radiation, Int. Rev. Cytol., 13 (1962) 22I 23I.
EVANS, H. J., Population cytogenetics and environmental factors, in Pfizer Medical 3/Ionographs 5, Edinburgh University Press, 197 ° , pp. 192-216. KIHLMAN, B. A., Actions of Chemicals on Dividing Cells, Prentice Hall, Englewood Cliffs, N.J., 1966.
