1、Designation: E 1280 97 (Reapproved 2003)Standard Guide forPerforming the Mouse Lymphoma Assay for MammalianCell Mutagenicity1This standard is issued under the fixed designation E 1280; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision
2、, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.INTRODUCTIONThis guide was developed at the request of ASTM Subcommittee E47.09 on Biomarkers in orderto aid toxico
3、logists, geneticists, biochemists, other researchers, and interested persons in theunderstanding, performance, and analysis of the mammalian cell mutagenicity test that uses theTK+/-3.7.2C strain of L5178Y mouse lymphoma cells. In this rapidly changing area of toxicology, itis not intended for this
4、guide to replace, alter, or diminish the usefulness of presently availableprotocols and procedures.1. Scope1.1 The purpose and scope of this guide is to presentbackground material and to establish criteria by which proto-cols and procedures for conducting the L5178Y/TK+/-3.7.2Cmouse lymphoma mutagen
5、icity assay (commonly referred to asthe mouse lymphoma assay, (MLA) can be properly under-stood and evaluated. This guide is also intended to aidresearchers and others to gain a better understanding of thecritical elements involved with mammalian cell mutagenicitytesting. More specifically, this gui
6、de is intended to provide forresearchers the accomplishment of the following goals:1.1.1 Provide an understanding of the critical procedures(steps) in the performance of this mammalian cell mutagenicitytest.1.1.2 Provide generalized criteria by which researchers canevaluate if they are properly perf
7、orming, utilizing, and inter-preting this assay.1.1.3 Provide criteria by which individuals responsible forevaluating MLA data can determine if the experiments havebeen properly performed and interpreted.1.1.4 Provide a basis from which new procedures anddevelopments in testing procedures can be eva
8、luated.1.1.5 Provide an understanding of the types of geneticdamage (that is, gene and chromosome mutation) that may bedetected in this mammalian cell mutagenicity test.2. Terminology2.1 Definitions:2.1.1 clastogenany agent that is capable of inducingchromosome breaks.2.1.2 gene mutationany heritabl
9、e change whose physicalextent is restricted to the limits of a single gene.2.1.3 mutagenany physical or chemical agent capable ofinducing a mutation.2.1.4 mutationany heritable change in the genetic mate-rial, not caused by genetic segregation or genetic recombina-tion, and that is transmitted to da
10、ughter cells.2.2 Definitions of Terms Specific to This Standard:2.2.1 chromosome mutationa mutation resulting from astructural change to a chromosome involving the gain, loss, orrelocation of chromosome segments. Chromosome mutationscan be either intrachromosomal or interchromosomal.2.2.2 relative s
11、uspension growth (RSG)used to measurethe cytotoxicity of a given treatment based on the growth ofcells in suspension culture relative to the untreated or solventcontrol(s). RSG is calculated according to the method of Cliveand Spector (1).22.2.3 relative total growth (RTG)used as a means tomeasure t
12、he relative toxicity to cells (survival) followingtreatment in the mouse lymphoma assay. RTG is calculatedaccording to the method of Clive and Spector (1) and includesRSG as well as the ability to form colonies in the clonal phaseof the assay.2.3 Symbols:2.3.1 BrUdR5-bromo-28-deoxyuridine.2.3.2 BUdR
13、bromouracil deoxyriboside.2.3.3 CASchemical abstract service.2.3.4 DMSOdimethylsulfoxide.2.3.5 MLAmouse lymphoma assay.1This guide is under the jurisdiction of Committee F04on Medical and SurgicalMaterials and Devices and is the direct responsibility of Subcommittee F04.16 onBiocompatibility Test Me
14、thods.Current edition approved Sept. 10, 2003. Published September 2003. Originallyapproved in 1989. Last previous edition approved in 1997 as E 1280 97.2The boldface numbers in parentheses refer to the list of references at the end ofthis guide.1Copyright ASTM International, 100 Barr Harbor Drive,
15、PO Box C700, West Conshohocken, PA 19428-2959, United States.2.3.6 NADPnicotinamide-adenine dinucleotide phos-phate.2.3.7 TFTtrifluorothymidine.2.3.8 THMGthymidine + hypoxanthine + methotrexate +glycine.2.3.9 VCviable count(s).3. Significance and Use3.1 This guide is limited to procedures used solel
16、y for thetesting of substances to determine their mutagenicity and doesnot apply to other methods and uses such as exploringmechanisms of mutation.3.2 Recent evidence suggests that this assay measures a dualgenetic end point; therefore, some discussion of the relation-ships between mammalian cell mu
17、tagenicity testing results andthe results observed both in pure gene mutational assays and incytogenetic assays is necessary. However, it is not the intent ofthis guide to discuss other relationships between this mamma-lian cell mutagenicity testing results and the results observed inother tests for
18、 mutagenicity and carcinogenicity.4. Test Materials4.1 MediaFischer(2) successfully adapted L5178Ymouse leukemic cells to growth in suspension culture usingF10 (Gibco H-11) medium. In developing and validating theL5178Y mouse lymphoma assay, Clive and associates (1)routinely used Fischers medium; ho
19、wever, other laboratorieshave recently validated the assay with RPMI 1640 medium(3-5). Either medium can be used; however, it is important tonote several differences between them. The most important ofthese is the large difference in phosphate concentration, a factorwhich can affect the stringency o
20、f trifluorothymidine (TFT)selection in RPMI medium (6) if proper precautions concern-ing heat inactivation and quality of horse serum are not taken(7); (see 4.1.4.1). Secondly, the effective concentrations ofcleansing medium components is dependent on the type of basemedium used (see 4.1.4.2). It is
21、 recommended that criticalcomponents (for example, horse serum) be heat-inactivatedeither separately or after combination. Fischers medium isphotosensitive in liquid formulations!4.1.1 Base MediumA base medium is generally preparedfrom powdered formulation or is purchased as a 103 or 13liquid. Some
22、laboratories prepare 23 medium which can beused for a variety of media preparations. Pluronic F683must beadded to the base medium to facilitate growth in suspensionculture. Other supplements usually include antibiotics, sodiumpyruvate, and occasionally, glutamine. Refer to references in4.1 for sugge
23、sted concentrations.4.1.2 Growth MediumGrowth medium is prepared bysupplementing the base medium with horse serum, usually10 % by volume.4.1.3 Cloning MediumCloning medium is growth me-dium further supplemented with agar (Noble, purified, orBaltimore Biological Laboratories (BBL); see Ref. (8) andof
24、ten with additional serum. Each investigator should deter-mine serum and agar concentrations that yield the best cloningconditions in their laboratory. See references in 4.1 for agar andserum concentrations as they vary between laboratories. Serumconcentration is often adjusted to 20 % in the clonin
25、g mediumsince this concentration has been reported to provide thehighest cloning efficiency for L5178Y cells (9); however, thisoptimum may vary among lots of horse serum and amonglaboratories.4.1.4 Selective MediaThere are two types of selectivemedia routinely used in the MLA: cloning medium supple-
26、mented with TFT to permit quantitation and characterization ofTK/mutants; and THMG cleansing medium which keeps thespontaneous TK/mutant frequency at a minimum, therebyoptimizing the assay sensitivity.4.1.4.1 TFT SelectionCloning medium supplementedwith TFT is used to arrest growth of TK+/cells and
27、to allowclonal growth of TK/cells. The optimal concentration of TFTmay vary among laboratories, but is usually in the range of 1to 5 g/ml. Those laboratories utilizing RPMI 1640 mediummay find it necessary to use a TFT concentration at the higherend of this range. Each laboratory should establish th
28、e efficacyof their TFT selection by appropriate means. Differential lots ofhorse serum vary in their ability to inactivate TFT, possiblyresulting from varying amounts of the enzyme thymidinephosphorylase. This enzyme, in the presence of inorganicphosphate, converts TFT to an inactive form. The appro
29、xi-mately sixfold higher level of inorganic phosphate present inRPMI 1640 medium (relative to Fischers medium) drives thisinactivation more rapidly in RPMI-based cloning medium ifthe serum is improperly heat inactivated, thereby criticallydecreasing TFT-selection stringency in the mutant selectionpl
30、ates. This can be overridden by a combination of increasedTFT concentration, extra attention to the proper heat inactiva-tion of the horse serum (that is, ensure that the serum reaches56C prior to initiating the 30 min incubation; Mayo, unpub-lished data) (2, 11), and stringent screening of serum lo
31、ts priorto routine use in the assay.NOTE 1Historically, 5-bromo-28-deoxyuridine (BUdR; BrUdR) hasbeen utilized with this assay to select for TK/cells. TFT has been shownto be a more effective selective agent, and the use of BUdR is discouraged(10).4.1.4.2 THMG CleansingCleansing medium (growth me-di
32、um supplemented with THMG) is one method used to rid thestock culture of spontaneously accumulated TK/mutants. Itis composed of: methotrexate (M), to block folate-dependentthymidylate synthase production of thymidine monophosphate(TMP), thus forcing the cells into dependency on the TKsalvage pathway
33、 of TMP synthesis; thymidine (T) and hypox-anthine (H), to bypass the folate block in TK-competent cells;and glycine (G) as a methyl group source. In TK/mutantcells, the exogenous thymidine cannot be phoshorylated, andthese cells die from TMP deficiency. Following 24-h growth inthe cleansing medium,
34、 the stock culture is centrifuged and thecells are washed free of unbound methotrexate and resus-pended in growth medium supplemented with THG (that is,THMG without methotrexate) for 1 to 3 days. This permits the3The sole source of supply of the apparatus known to the committee at this timeis BASF W
35、yandotte Corp., Wyandotte, MI 48192. If you are aware of alternativesuppliers, please provide this information to ASTM International Headquarters.Your comments will receive careful consideration at a meeting of the responsibletechnical committee,1which you may attend.E 1280 97 (2003)2cells to fully
36、recover from the remaining bound methotrexateand resume synthesis of TMP and purines by the folate-dependent pathways. Cells should be allowed to totally recoverfrom the metabolic stress of the cleansing procedure (about 2to 3 days) before being used in a test.4.1.4.3 While it has been suggested tha
37、t the cleansingprocedure be performed on a weekly basis, some laboratoriesmay find a less frequent cleansing schedule acceptable, pro-viding a low background mutation frequency is maintained.Other alternatives include: freezing populations of freshlycleansed cells and thawing them a few days prior t
38、o use; usingcultures grown for a very low inoculum (ca. 600 cells/culture;however, this method suffers from potential genetic driftproblems which could alter this well-characterized cell line); ormaintaining an uncleansed population of cells and cleansing aportion of these cells prior to use. In the
39、se cases the exposureof TK+/cells to methotrexate, which, in the absence of THGis known to induce mutations, can be reduced to a minimum.For specific concentrations of the ingredients and cell popula-tions used in the cleansing step, refer to references in 4.1. It isimportant to note for those labor
40、atories utilizing RPMI 1640medium, that slightly higher concentrations of THMG andTHG are required, as noted in the literature.4.1.5 Quality Control of MediaThe quality of culturemedia is a common cause of problems with the MLA. Anumber of factors are known to contribute to variations inmedium quali
41、ty, the principal ones being water quality andexposure of liquid Fischers medium to excessive light. An-other identified source of assay problems is the lot and sourceof agar (8) and the problem of the use of a dirty autoclave tosterilize the agar. Serum requires particular precautions withRPMI medi
42、um (7, 11); see 4.1.4.1. For these reasons, rigorousmethods for media quality control should be established foreach laboratory to address the ability to support: (1) suspensiongrowth of both low (#1000 cells/mL) and high (1 3 106cells/mL) cell inocula, (2) high cloning efficiencies undernonselective
43、 conditions, (3) adequate recoveries of small andlarge colony TK/mutants, and (4) appropriate diameters ofnonmutant and both classes of mutant colonies. Each of thesequantities should be consistent with published literature values.4.2 Metabolic Activation SystemThe metabolic activationsystem may tak
44、e the form of either whole cells (for example,cocultivated rat hepatocytes (12 and 13) or cell homogenates(for example, Aroclor-1254induced rat liver S9 (14); Aroclor-1254induced hamster or mouse S9 (15).4.2.1 SourcesPreparations designed to provide metabolicactivation may be prepared from a variety
45、 of sources depend-ing on the needs of a particular assay. Factors which may varyinclude, for example, species, sex, tissue, age, method ofinduction, and method of preparation.4.2.2 Cofactor Mixes for Enzyme Preparationsshould beshown to support enzyme activity, as measured either directlyor by a bi
46、ological effect. Commonly used cofactors includeNADP in conjunction with either sodium isocitrate or glucose-6-phosphate (3, 5, 14-16).4.2.3 Metabolic ActivitiesThe metabolic activation sys-tem to be used should be capable of converting appropriateknown promutagens to mutagens while causing little o
47、r notoxicity or mutagenicity to the mouse lymphoma cells in thesolvent control culture(s).5. Test Method5.1 Test PrincipleThe mouse lymphoma assay utilizes astrain (TK+/-3.7.2C clonal line) of L5178Y mouse lymphomacells that has been made heterozygous at the TK locus (17).These cells contain the TK
48、enzyme and are sensitive to thecytostatic and cytotoxic effects of appropriate concentrations ofTFT (10). Forward mutations to the single functional TK genecan result in the loss of TK activity and thus the acquisition ofTFT-resistance. These mutant cells can be quantitated after anappropriate expre
49、ssion period by cloning in a soft agar mediumsupplemented with the selective agent, TFT (10, 18). A numberof protocols have been described (1, 14-19). The assay hasbeen adapted to detect a wide variety of mutagens includingthose requiring exogenous metabolic activation.5.2 Description of Test System:5.2.1 Cell LineThe MLA uses the TK+/-3.7.2C heterozy-gote of L5178Y mouse lymphoma cells (17). This cell line hasbeen cytogenetically characterized by banded karyotype at the230 to 300-band level of resolution (20 and 21). The
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