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hypoxanthine-guanine phosphoribosyl transferase gene mutation assay
Mutation Research, 189 (1987) 135-141
135
Elsevier
M T R 01171
A guide for the p e r f o r m a n c e of the C h i n e s e h a m s t e r ovary c e l l / h y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l transferase gene m u t a t i o n assay A.P. Li 1 J.H. Carver 2 W.N. Choy 3 A.W. Hsie 4 R.S. Gupta 5, K.S. Loveday 6 J.P. O'Neill 7, J.C. Riddle 8, L.F. Stankowski Jr. 9 and L.L. Yang 10 ,
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9
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I Environmental Health Laboratory, Monsanto Company, St. Louis, MO 63167 (U.S.A.), 2 Chevron Environmental Health Center Inc., Richmond, CA 94804 (U.S.A.), 3 Haskell Laboratory for Toxicology and Industrial Medicine, E.L Du Pont de Nemours and Co., Newark, DE 19711 (U.S.A.), 4 Health and Safety Division and Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 and Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.), 5 Department of Biochemistry, McMaster University, Hamilton, Ont. LSN 3Z5 (Canada), 6 American Biogenics Corporation, Woburn, MA 01801 (U.S.A.), z Genetics Laboratory, Vermont Regional Cancer Center, Burlington, VT 05401 (U.S.A.), s B.F. Goodrich Research and Development Center, Brecksville, OH 44141 (U.S.A.), 9 Pharmakon Research International, Inc., Waverly, PA 18471 (U.S.A.)and lo Microbiological Associates, Inc., Bethesda, MD 20816 (U.S.A.) (Received 13 January 1987) (Accepted 16 January 1987)
Keywords: Guide; Gene mutation assay; Chinese hamster ovary cell; H y p o x a n t h i n e - g u a n i n e phosphoribosyl transferase; C H O / H G P R T assay; Mammalian cells; Cell culture.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assay description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of C H O cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenesis procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Mutagen treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a) Cell plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (b) Chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (c) Addition of test article to cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (d) Exogenous activation systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (e) Estimation of cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (f) Positive and solvent controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Expression of induced mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Mutant selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Criteria for data acceptability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Testing strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Correspondence: Dr. A.P. Li, Monsanto Company, Mail Zone EHL, 800 N. Lindbergh, St. Louis, M O 63167 (U.S.A.). 0165-1218/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
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Introduction
The Chinese hamster ovary c e l l / h y p o x a n thine-guanine phosphoribosyl transferase ( C H O / H G P R T ) assay (Hsie et al., 1981) has been widely applied to the toxicological evaluation of industrial and environmental chemicals. This document represents our effort to provide a set of general guidelines for the performance of the assay. It is intended to highlight some of the more relevant biological concepts as they are currently understood, and to summarize the critical technical aspects for acceptable bioassay performance as they are currently perceived and practiced. This document concentrates on the practical aspects of cell culture, mutagenesis procedures, data analysis, quality control and testing strategy. The suggested approach represents a consensus of the panel members* for the performance of the assay. It is to be understood, however, that these are merely general guidelines and are not to be followed without the use of sound scientific judgement. Users of the assay should evaluate their approach based on the properties of the substances to be tested and the questions to be answered. Deviation from our guidelines based on sound scientific judgement should by no means invalidate the results obtained. Assay description The C H O / H G P R T assay detects forward mutations of the X-linked hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus (coding for the enzyme, H G P R T ) in Chinese hamster ovary (CHO) cells. Cells originally derived from Chinese hamster ovary tissue are exposed to a test article and, following an appropriate cell culture regimen, descendants of the original treated population are monitored for the loss of functional H G P R T , presumably due to mutations. Resistance to a purine analogue, 6-thioguanine (6TG) (or less desirably, 8-azaguanine (8AG)), is employed as the * The authors of this manuscript belong to the CHO/HGPRT panel of the Genetic Toxicology Subcommittee of the American Societyof Testing and Materials (Chairman, A.P. El).
genetic marker. H G P R T catalyzes the conversion of the nontoxic 6TG to its toxic ribophosphorylated derivative. Loss of the enzyme or its activity therefore leads to cells resistant to 6TG. Because H G P R T is an enzyme of the purine nucleotide salvage pathway, loss of the enzyme is not a lethal event. Different types of mutational events (base substitutions, frameshifts, deletions, some chromosomal type lesions, etc.) should theoretically be detected at the hgprt locus. The C H O / H G P R T assay has been used to study a wide range of mutagens, including radiations (Hsie et al., 1977; Riddle and Hsie, 1978; Yuhas et al., 1979), a wide variety of chemicals (Hsie et al., 1981), and complex chemical mixtures (Li et al., 1983). Characteristics of C H O cells
Different CHO cell lines/subclones are appropriate for the C H O / H G P R T assay. The C H O - K : B H 4 cell line developed and extensively characterized by Hsie et al. (1975, 1979) is probably the most widely employed. The CHO(WT) cell line and its derivative, CHO-AT3-2, are used to monitor mutations at other gene loci in additional to hgprt (Gupta and Siminovitch, 1980; Carver et al., 1980). While there are differences among the cell lines employed, a number of general characteristics are critical for the performance of the assay: (1) The cloning efficiency (CE) of the stock cultures should not be less than 70%. The CE of untreated experimental cultures should not be less than 50%. (2) Cultures in logarithmic phase of growth should have a population doubling time of 12-16 h. (3) The modal chromosome number should be 20 or 21, as is characteristic of the particular subclone used. (4) Cultures should be free of microbial and mycoplasma contamination. These cell properties that are critical for the assay should be routinely monitored as part of the quality control regimen. Routine quality control procedures should include testing of serum and media for each new purchase, as well as mycoplasma and karyotype checks at least once yearly.
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Mutagenesis procedures The mutagenesis protocol can be divided into 3 phases: mutagen treatment, expression and selection.
Mutagen treatment (a) Cellplating. Cells should be in exponential phase when plated for treatment. Several media (e.g. F12, alpha-MEM) that are known to be optimal for cell growth can be used. Cells should be seeded at an appropriate cell density to allow exponential growth as well as quantitation of induced responses. A common practice is to plate 0.5 x 106 cells in a 25-cm2 flask, or 1.5 x 106 cells in a 75-cm2 flask, on the day before treatment. (b) Chemical handling. The solubility of the test article in an appropriate medium should be determined before treatment. Commonly used solvents are, in the order of preference, medium, water, dimethyl sulfoxide, ethanol and acetone. Generally, the nonaqueous solvent concentration should not exceed 1% and should be constant for all samples. As part of the solubility test, an aliquot of the test chemical should be added to the treatment medium to note any pH changes, the presence of any chemical precipitation, and any apparent reaction of the chemical or' solvent with the culture vessel. The solvent of choice should not have any undesirable reactions with the test article, culture vessel or cells. (c) Addition of test article to cells. Stock solutions of the test samples are prepared and aliquots added to each flask. Dilutions of the test article should be such that the concentration of solvent remains constant for all samples. Cells are generally treated with the test article for at least 3 h. For treatment times of 3-5 h, serum-free medium can be used. As serum is required to maintain cell division, medium containing serum should be used for a prolonged treatment period (e.g. 16 h or longer). Serum requirement for treatment periods between 5 and 16 h should be determined on a case-by-case basis. (d) Exogenous activation systems. Aroclor 1254-induced rat-liver homogenate ($9) is the most
commonly used exogenous metabolic activating system for the assay. When $9 is used, cofactors for the mixed function monooxygenases should be present. Calcium chloride, which enhances the mutagenicity of nitrosamines and polycyclic hydrocarbons (Machanoff et al., 1981; Li, 1984), appears to be another useful addition. However, the need for CaC12 has yet to be documented for a wide variety of chemicals. A commonly used cofactor mixture consists of sodium phosphate (50 mM, pH 7.0-8.0), N A D P (4 mM), glucose 6-phosphate (5 mM), KC1 (30 mM), MgC12 (10 mM) and CaC12 (10 mM). $9 is added directly to the cofactor mixture. 1 vol. of the cofactor mixture is added to 4 vol. of treatment medium. Other exogenous systems (e.g. hepatocytes, $9 from other animal species or produced using different enzyme induction conditions, and other cofactor mixtures) can also be used depending on the intent of the experiment.
(e) Estimation of cytotoxicity. Plating CHO cells immediately after treatment for cytotoxicity determination is generally expected to yield the most accurate results. Otherwise, cytotoxicity can be estimated on the day after treatment. Aliquots of the cells are plated to allow for colony development. Cytotoxicity is usually expressed as relative CE which is the ratio of the CE of the treated cells to that of the solvent control. Viability determination should take into account any loss of cells during the treatment period, cell trypsinization procedures, a n d / o r the overnight incubation period. (f) Positive and solvent controls. An appropriate solvent control is treatment of cells with the solvent used for the test article. Positive controls, both direct-acting and indirect-acting, should also be included to demonstrate the adequacy of the experimental conditions to detect known mutagens. An untreated control may also be included to evaluate the effects of the solvent on mutagenicity. Commonly used positive controls are ethyl methanesulfonate (EMS) and N-methylN'-nitro-N-nitrosoguanidine ( M N N G ) as directacting mutagens, and benzo[a]pyrene (BaP) and dimethylnitrosamine (DMN) as promutagens that require metabolic activation.
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(2) Expression of induced mutations After mutation at the hgprt locus, the mutant phenotype requires a period of time before it is completely expressed (expression requires the loss of pre-existing enzyme activity). Phenotypic expression is presumably achieved by dilution of the pre-existing H G P R T enzyme and m R N A through cell division and macromolecular turnover. At the normal population doubling times of 12-16 h for CHO cells, an expression period of 7 - 9 days is generally adequate (O'Neill et al., 1977; O'Neill and Hsie, 1979a). The most widely employed method for phenotypic expression allows exponential growth of the cells for a defined time period after mutagen treatment. CHO cells can be subcultured with 0.05% trypsin with or without EDTA. Aliquots of 1 x 106 cells are subcultured at 2- or 3-day intervals in 100-mm diameter tissue culture dishes or 75-cm2 t-flasks. Either complete medium or hypoxanthine-free medium can be employed, with either dialyzed or nondialyzed serum. It is important to ensure that the medium employed will allow a population doubling time of 12-16 h. Besides the normal growth of cells as monolayer cultures, alternative methods of subculturing involving suspension (Carver et al., 1980), unattached (Li, 1981), and division-arrested (O'Neill, 1982) cultures have also been successful. The use of a particular subculture regimen in the expression period should be substantiated by data demonstrating the achievement of optimal expression.
(3) Mutant selection Conditions for the selection of mutants must be defined to ensure that only mutant cells are able to form colonies and that all mutant cells form colonies. In general, cells are plated in tissue culture dishes for attached colony growth (O'Neill et al., 1977; O'Neill et al., 1977), or in agar for suspended colony growth (Li and Shimizu, 1983). An advantage of the former is that after the colonies are fixed and stained, the plates can be counted at a later date. An advantage of the latter is that metabolic cooperation between wild-type and mutant cells is reduced, allowing selection of a higher cell number per plate. Reliable selection has been established in hypoxanthine-free medium containing dialyzed serum
and 10 ~M 6TG. Fetal bovine serum, newborn bovine serum, or calf serum can be used, providing that the serum has been adequately tested and shown to support the desirable characteristics of CHO cells as described above. Dialyzed serum is usually necessary to eliminate the competition between 6TG and purine bases in the serum. It has been found that a selection cell density of 2 x 105 or fewer cells per 100-mm dish for attached colony growth (O'Neill et al., 1977; O'Neill and Bowman, 1982) and 10 6 o r fewer cells per 100-mm dish (in 30 ml agar) for agar colony growth (Li and Shimizu, 1983) allows essentially 100% recovery of mutant cells.
Data presentation Results from the assay should include the following experimental data: (1) Concentrations and solvents used for the test article and positive controls. (2) Absolute and relative cloning efficiencies (CE) in the concurrent cytotoxicity assay, where Absolute CE -
number of coloniesformed number of cells plated
Relative CE =
CE (treatment) CE (solventcontrol)
(3) Actual number of mutant colonies observed for each treatment condition. (4) Absolute CE at selection for each treatment condition. (5) Mutant frequency (MF) values, where MF=
number of mutant colonies number of clonable cells
and Number of colonablecells = number of cells plated × absolute CE at selection MF is usually expressed as mutants per clonable cells.
10 6
Criteria for data acceptability Generally, for the data of a given assay to be acceptable, the following criteria should be met:
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(1) The absolute CE of the negative controls should not be less than 50%. Absolute CE values lower than 50% would indicate suboptimal culturing conditions for the cells. (2) The mean mutant frequency of the solvent controls in each experiment should fall within the range of 0-20 mutants per 1 0 6 clonable cells. A higher mutant frequency may preclude detection of weak mutagens. Under such conditions data acceptability should be evaluated on a case by case basis. (3) The positive control must induce a statistically significant response at a magnitude appropriate for the mutagen under the chosen experimental conditions. (4) The highest test article concentration should, if possible, result in a significant cytotoxic response (e.g. 10-30% survival, where survival is the percent of the treated population that is viable after treatment). This is particularly important if the response is negative. For noncytotoxic test articles, the highest concentration has generally been 1-10 mg per ml, or to the limit of solubility.
Data analysis Due to possibility of fluctuation, only samples with fewer than 105 viable cells after treatment should be used with caution for data analysis. Judgement on mutagenicity should be made based on the following: (1) Dose-reponse relationship. (2) Significance of response (in comparison to the negative control). (3) Reproducibility of the results. Exact statistical analysis is difficult because the distribution of the number of mutant colonies depends on the complex processes of cell growth and death after mutagen treatment. While other appropriate methods can be used, two commonly used approximate methods are as follows: (1) A weighted regression analysis where the weights are proportional to the observed number of mutant colonies divided by the square of the observed mutant frequency (Hsie et al., 1975). This weighting scheme was derived by assuming that the variance of the observed mutant frequency is a constant multiple of that which would occur if the number of mutant colonies on each selection
plate has a Poisson distribution. A test compound is considered to exhibit a mutagenic response if the slope of the mutant induction as a function of test concentrations is greater than zero at the 0.01 level according to the t-test (Tan and Hsie, 1981). (2) A power transformation procedure with which the observed mutant frequency is transformed using the formula Y = ( X + A ) B, w h e r e Y = transformed mutant frequency X = o b s e r v e d m u t a n t f r e q u e n c y , and A, B = c o n s t a n t s
Data transformed by this method appears to satisfy the assumptions of homogeneous variance and normal distribution (Snee and Irr, 1981). Comparison to negative control values and doseresponse relationships are examined with Student's t-test and an analysis of variance (ANOVA) using the transformed values. Computations can be done with computer programs readily available.
Testing strategy In general, the mutagenicity test should b e designed to consider the following: (1) The test substance should be tested at levels allowing significant chemical-cell interaction, which is generally indicated by cytotoxicity: Relatively insoluble chemicals should be tested to the limit of solubility. Nontoxic but highly soluble chemicals should be tested to an arbitrary maximum concentration based on the anticipated human exposure level and a conservative safety factor. As a general rule of thumb, 1-10 m g / m l should be sufficient as the maximum concentration. (2) Different amounts of Aroclor 1254-induced liver $9 may be used, since it has been shown that some mutagens may be highly sensitive to the level of $9 used (Machanoff et al., 1981; Li, 1984). (3) The observation should be reproducible as indicated by two or more independent experiments. (4) In each experiment, intra-experimental variations should be determined using replicate treatment cultures. An example of an adequate combination of experiments (Li, 1985) is as follows: ( a ) Expt. 1. Range-finding for cytotoxicity. Log
140 or half-log concentrations of the test articles are evaluated in the absence and presence of various levels of $9. Cytotoxicity information obtained is used for dose selection in the subsequent m u t a genesis experiments. A repeat of the experiment using a narrower concentration range m a y be necessary for test articles with steep cytotoxic responses.
( b ) Expt. 2. Initial mutagenicity determination with limited doses and at multiple $9 concentrations. This experiment should yield information for an initial estimation of mutagenicity as well as any effects of $9 concentration on mutagenicity. Concentrations of the test article are selected based on the results of Expt. 1.
(c) Expt. 3. Confirmatory mutagenicity determination. This experiment would incorporate the optimal $9 level, if any, as determined in Expt. 2. A larger n u m b e r of concentrations than in Expt. 2 should be used for a more accurate estimation of the d o s e - r e s p o n s e relationship. General guidelines for the performance of this assay for chemical testing have also been published, and can be used as a basis for experimental design (e.g. O'Neill and Hsie, 1979b; Hsie et al., 1981; EPA Health Effects Test Guidelines, 1982; O E C D , 1983). Other considerations Based on the latest scientific information and practical experience, we have developed the above guidelines for the performance of the C H O / H G P R T assay. We c a n n o t overemphasize the fact that these are merely guidelines. This d o c u m e n t should not be viewed as encompassing the only available, appropriate or useful protocols and procedures. There is no substitute for sound scientific judgement and " h a n d s o n " experience. This document, therefore, should not be construed as an instrument for inhibiting present or future research and development towards further refinem e n t of the assay. Being an extensively characterized assay, the CHO/HGPRT assay should be useful in the toxicological evaluation of industrial and environmental substances. A n advantage of using C H O cells is that other well-characterized genotoxicity endpoints have also been developed, e.g., muta-
tions at other gene loci (Carver et al., 1980; G u p t a and Siminovitch, 1980; G u p t a and Singh, 1982; Singh and Gupta, 1983a; Sing and Gupta, 1983b), c h r o m o s o m a l aberrations (Preston et al., 1981), and sister-chromatid-exchanges (Wolff, 1977). It is therefore possible to use a variety of endpoints in C H O cells for testing, yielding additional information that m a y be used, in conjunction with data from other toxicity assays, for the prediction of the h u m a n toxicological consequences of exposure to the substances tested. Acknowledgement The authors are grateful for the effort of Dr. Robert Naismith who, as a chariman of the A S T M subcommittee on Genetic Toxicology, has been instrumental for the initiation of the C H O / H G P R T work group. References Carver, J.H., G.M. Adair, and D.L. Wandres (1980) Mutagenicity testing in mammalian cells, II. Validation of multiple drug resistance markers having practical application for screening potential mutagens, Mutation Res., 72, 207-230. EPA Health Effects Test Guidelines (1982) EPA 560/6-82-001. Gupta, R.S., and L. Siminovitch (1980) Genetic markers for quantitative mutagenesis studies in Chinese hamster ovary ceils: Characteristics of some recently developed selective systems, Mutation Res., 69, 113-126. Gupta, R.S., and B. Singh (1982) Mutagenic response in five independent genetic locus in CHO cells to a variety of mutagens: Development and characteristics of a mutagen screening system based on selection for multiple drug resistant markers, Mutation Res., 94, 449-466. Hsie, A.W., P.A. Brimer, T.J. Mitchell and D.G. Gosslee (1975) The dose-response relationships for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells, Somatic Cell Genet., 1, 247-261. Hsie, A.W., A.P. Li and R. Machanoff (1977) A fluence-response study of lethality and mutagenicity of white, black, and blue florescent light, sunlamp, and sunlight irradiation in Chinse hamster ovary cells, Mutation Res., 45, 333-342. Hsie, A.W., J.P. O'Neill, D.B. Couch, J.R. SanSebastian, P.A. Brimer, R. Machanoff, J.C. Riddle, A.P. Li, J.C. Fuscoe, N.L. Forbes and M.H. Hsie (1979) Utilization of a quantitative mammalian cell mutation system in experimental mutagenesis and genetic toxicology, in: B. Butterworth (Ed.), Strategy for Short-Term Testing for Mutagens/ Carcinogens, C.R.C. Press, West Palm Beach, pp. 39-54. Hsie, A.W., D.A. Casiano, D.B. Couch, D.F. Krahn, J.P. O'Neill and B.L. Whitfield (1981) The use of Chinese hamster ovary cells to quantify specific locus mutation and
141 to determine mutagenicity of chemicals, A report of the Gene-Tox program, Mutation Res., 86, 193-224. Li, A.P. (1981) Simplification of the C H O / H G P R T mutation assay through the growth of Chinese hamster ovary cells as unattached cultures, Mutation Res., 85, 165-175. Li, A.P. (1984) Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells, Environ. Mutagen., 6, 539-544. Li, A.P. (1985) A testing strategy to evaluate the mutagenic activity of industrial chemicals in cultured mammalian cells, Regul. Toxicol. Pharmacol., 5, 207-211. Li, A.P., and R.W. Shimizu (1983) A modified agar assay for the quantitation of mutation at the hypoxanthine guanine phosphoribosyl transferase gene locus in Chinese hamster ovary cells, Mutation Res., 111, 365-370. Li, A.P., A.L. Brooks, C.R. Clark, R.W. Shimizu, R.L Hansen and J.S. Dutcher (1983) Mutagenicity testing of complex environmental mixtures with Chinese hamster ovary cells, in: M. Waters, S. Sandbu, J. Lewtas, L. Claxton, M. Chernoff and S. Nesnow (Eds.), Short-term Bioassays in the Analysis of Complex Environmental Mixtures III, Plenum, New York, pp. 183-196. Machanoff, R., J.P. O'Neill and A.W. Hsie (1981) Quantitative analysis of cytotoxicity and mutagenicity of benzo(a)pyrene in mammalian cells ( C H O / H G P R T system), Chem.-Biol. Interact., 34, 1-10. OECD (1983) Guidelines for Testing of Chemicals: Genetic Toxicology. In vitro Mammalian Cell Gene Mutation Tests (final draft). O'Neill, J.P. (1982) Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division, Mutation Res., 106, 113-122. O'Neill, J.P., and K.O. Bowman (1982) Effect of selection cell density on the recovery of mutagen induced TG r cells, Environ. Mutagen., 4, 627-637. O'Neill, J.P., and A.W. Hsie (1979a) Phenotypic expression time of mutagen induced 6-thioguanine resistance in CHO ceils, Mutation Res., 59, 109-118. O'Neill, J.P., and A.W. Hsie (1979b) C H O / H G P R T Mutation
Assay: Experimental Procedure, in: A.W. Hsie, J.P. O'Neill and V.K. McElheny (Eds.), Banbury Report 2, Mammalian Cell Mutagenesis: The Maturation of Test Systems, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 55-69. O'Neill, J.P., P.A. Brirner, R. Machanoff, G.P. Hirsch and A.W. Hsie (1977) A quantitative assay of mutation induction at the hgprt locus in CHO cells, Mutation Res., 45, 91-101. Preston, R.J., W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McFee, S. Wolff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenesis assay: A report of the U.S. Gene-Tox program, Mutation Res., 87, 143-188. Riddle, J.C., and A.W. Hsie (1978) An effect of cell cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells, Mutation Res., 52, 409-420. Singh, B., and R.S. Gupta (1983a) Comparison of the mutagenic responses of twelve anticancer drugs at the hypoxanthine-guanine phosphoribosyl transferase and adenosine kinase loci in Chinese hamster ovary cells, Environ. Mutagen., 5, 871-880. Singh, B., and R.S. Gupta (1983b) Mutagenic responses of thirteen anticancer drugs on mutation induction at multiple genetic loci and on sister chromatid exchanges in Chinese hamster ovary cells, Cancer Res., 43, 577-584. Snee, R.D., and J.D. Irr (1981) Design of a statistical method for the analysis of mutagenesis at the hypoxanthine-gnanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells, Mutation Res., 85, 77-93. Tan, E.-L., and A.W. Hsie (1981) Mutagenicity and cytotoxicity of haloethanes as studied in the C H O / H G P R T system, Mutation Res., 90, 183-191. Wolff, S. (1977) Sister chromatid exchange, Annu. Rev. Genet., 11, 183-201. Yuhas, J.M., A.P. Li and M.N. Kligerman (1979) Present status of the proposed of negative pimesons in radiotherapy, in: J.T. Lett and H. Adler (Eds.), Advances in Radiation Biology, Vol. 8, Academic Press, New York, pp. 51-85.
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Elsevier
M T R 01171
A guide for the p e r f o r m a n c e of the C h i n e s e h a m s t e r ovary c e l l / h y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l transferase gene m u t a t i o n assay A.P. Li 1 J.H. Carver 2 W.N. Choy 3 A.W. Hsie 4 R.S. Gupta 5, K.S. Loveday 6 J.P. O'Neill 7, J.C. Riddle 8, L.F. Stankowski Jr. 9 and L.L. Yang 10 ,
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9
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I Environmental Health Laboratory, Monsanto Company, St. Louis, MO 63167 (U.S.A.), 2 Chevron Environmental Health Center Inc., Richmond, CA 94804 (U.S.A.), 3 Haskell Laboratory for Toxicology and Industrial Medicine, E.L Du Pont de Nemours and Co., Newark, DE 19711 (U.S.A.), 4 Health and Safety Division and Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 and Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.), 5 Department of Biochemistry, McMaster University, Hamilton, Ont. LSN 3Z5 (Canada), 6 American Biogenics Corporation, Woburn, MA 01801 (U.S.A.), z Genetics Laboratory, Vermont Regional Cancer Center, Burlington, VT 05401 (U.S.A.), s B.F. Goodrich Research and Development Center, Brecksville, OH 44141 (U.S.A.), 9 Pharmakon Research International, Inc., Waverly, PA 18471 (U.S.A.)and lo Microbiological Associates, Inc., Bethesda, MD 20816 (U.S.A.) (Received 13 January 1987) (Accepted 16 January 1987)
Keywords: Guide; Gene mutation assay; Chinese hamster ovary cell; H y p o x a n t h i n e - g u a n i n e phosphoribosyl transferase; C H O / H G P R T assay; Mammalian cells; Cell culture.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assay description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of C H O cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenesis procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Mutagen treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a) Cell plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (b) Chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (c) Addition of test article to cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (d) Exogenous activation systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (e) Estimation of cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (f) Positive and solvent controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Expression of induced mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Mutant selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Criteria for data acceptability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Testing strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Correspondence: Dr. A.P. Li, Monsanto Company, Mail Zone EHL, 800 N. Lindbergh, St. Louis, M O 63167 (U.S.A.). 0165-1218/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
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Introduction
The Chinese hamster ovary c e l l / h y p o x a n thine-guanine phosphoribosyl transferase ( C H O / H G P R T ) assay (Hsie et al., 1981) has been widely applied to the toxicological evaluation of industrial and environmental chemicals. This document represents our effort to provide a set of general guidelines for the performance of the assay. It is intended to highlight some of the more relevant biological concepts as they are currently understood, and to summarize the critical technical aspects for acceptable bioassay performance as they are currently perceived and practiced. This document concentrates on the practical aspects of cell culture, mutagenesis procedures, data analysis, quality control and testing strategy. The suggested approach represents a consensus of the panel members* for the performance of the assay. It is to be understood, however, that these are merely general guidelines and are not to be followed without the use of sound scientific judgement. Users of the assay should evaluate their approach based on the properties of the substances to be tested and the questions to be answered. Deviation from our guidelines based on sound scientific judgement should by no means invalidate the results obtained. Assay description The C H O / H G P R T assay detects forward mutations of the X-linked hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus (coding for the enzyme, H G P R T ) in Chinese hamster ovary (CHO) cells. Cells originally derived from Chinese hamster ovary tissue are exposed to a test article and, following an appropriate cell culture regimen, descendants of the original treated population are monitored for the loss of functional H G P R T , presumably due to mutations. Resistance to a purine analogue, 6-thioguanine (6TG) (or less desirably, 8-azaguanine (8AG)), is employed as the * The authors of this manuscript belong to the CHO/HGPRT panel of the Genetic Toxicology Subcommittee of the American Societyof Testing and Materials (Chairman, A.P. El).
genetic marker. H G P R T catalyzes the conversion of the nontoxic 6TG to its toxic ribophosphorylated derivative. Loss of the enzyme or its activity therefore leads to cells resistant to 6TG. Because H G P R T is an enzyme of the purine nucleotide salvage pathway, loss of the enzyme is not a lethal event. Different types of mutational events (base substitutions, frameshifts, deletions, some chromosomal type lesions, etc.) should theoretically be detected at the hgprt locus. The C H O / H G P R T assay has been used to study a wide range of mutagens, including radiations (Hsie et al., 1977; Riddle and Hsie, 1978; Yuhas et al., 1979), a wide variety of chemicals (Hsie et al., 1981), and complex chemical mixtures (Li et al., 1983). Characteristics of C H O cells
Different CHO cell lines/subclones are appropriate for the C H O / H G P R T assay. The C H O - K : B H 4 cell line developed and extensively characterized by Hsie et al. (1975, 1979) is probably the most widely employed. The CHO(WT) cell line and its derivative, CHO-AT3-2, are used to monitor mutations at other gene loci in additional to hgprt (Gupta and Siminovitch, 1980; Carver et al., 1980). While there are differences among the cell lines employed, a number of general characteristics are critical for the performance of the assay: (1) The cloning efficiency (CE) of the stock cultures should not be less than 70%. The CE of untreated experimental cultures should not be less than 50%. (2) Cultures in logarithmic phase of growth should have a population doubling time of 12-16 h. (3) The modal chromosome number should be 20 or 21, as is characteristic of the particular subclone used. (4) Cultures should be free of microbial and mycoplasma contamination. These cell properties that are critical for the assay should be routinely monitored as part of the quality control regimen. Routine quality control procedures should include testing of serum and media for each new purchase, as well as mycoplasma and karyotype checks at least once yearly.
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Mutagenesis procedures The mutagenesis protocol can be divided into 3 phases: mutagen treatment, expression and selection.
Mutagen treatment (a) Cellplating. Cells should be in exponential phase when plated for treatment. Several media (e.g. F12, alpha-MEM) that are known to be optimal for cell growth can be used. Cells should be seeded at an appropriate cell density to allow exponential growth as well as quantitation of induced responses. A common practice is to plate 0.5 x 106 cells in a 25-cm2 flask, or 1.5 x 106 cells in a 75-cm2 flask, on the day before treatment. (b) Chemical handling. The solubility of the test article in an appropriate medium should be determined before treatment. Commonly used solvents are, in the order of preference, medium, water, dimethyl sulfoxide, ethanol and acetone. Generally, the nonaqueous solvent concentration should not exceed 1% and should be constant for all samples. As part of the solubility test, an aliquot of the test chemical should be added to the treatment medium to note any pH changes, the presence of any chemical precipitation, and any apparent reaction of the chemical or' solvent with the culture vessel. The solvent of choice should not have any undesirable reactions with the test article, culture vessel or cells. (c) Addition of test article to cells. Stock solutions of the test samples are prepared and aliquots added to each flask. Dilutions of the test article should be such that the concentration of solvent remains constant for all samples. Cells are generally treated with the test article for at least 3 h. For treatment times of 3-5 h, serum-free medium can be used. As serum is required to maintain cell division, medium containing serum should be used for a prolonged treatment period (e.g. 16 h or longer). Serum requirement for treatment periods between 5 and 16 h should be determined on a case-by-case basis. (d) Exogenous activation systems. Aroclor 1254-induced rat-liver homogenate ($9) is the most
commonly used exogenous metabolic activating system for the assay. When $9 is used, cofactors for the mixed function monooxygenases should be present. Calcium chloride, which enhances the mutagenicity of nitrosamines and polycyclic hydrocarbons (Machanoff et al., 1981; Li, 1984), appears to be another useful addition. However, the need for CaC12 has yet to be documented for a wide variety of chemicals. A commonly used cofactor mixture consists of sodium phosphate (50 mM, pH 7.0-8.0), N A D P (4 mM), glucose 6-phosphate (5 mM), KC1 (30 mM), MgC12 (10 mM) and CaC12 (10 mM). $9 is added directly to the cofactor mixture. 1 vol. of the cofactor mixture is added to 4 vol. of treatment medium. Other exogenous systems (e.g. hepatocytes, $9 from other animal species or produced using different enzyme induction conditions, and other cofactor mixtures) can also be used depending on the intent of the experiment.
(e) Estimation of cytotoxicity. Plating CHO cells immediately after treatment for cytotoxicity determination is generally expected to yield the most accurate results. Otherwise, cytotoxicity can be estimated on the day after treatment. Aliquots of the cells are plated to allow for colony development. Cytotoxicity is usually expressed as relative CE which is the ratio of the CE of the treated cells to that of the solvent control. Viability determination should take into account any loss of cells during the treatment period, cell trypsinization procedures, a n d / o r the overnight incubation period. (f) Positive and solvent controls. An appropriate solvent control is treatment of cells with the solvent used for the test article. Positive controls, both direct-acting and indirect-acting, should also be included to demonstrate the adequacy of the experimental conditions to detect known mutagens. An untreated control may also be included to evaluate the effects of the solvent on mutagenicity. Commonly used positive controls are ethyl methanesulfonate (EMS) and N-methylN'-nitro-N-nitrosoguanidine ( M N N G ) as directacting mutagens, and benzo[a]pyrene (BaP) and dimethylnitrosamine (DMN) as promutagens that require metabolic activation.
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(2) Expression of induced mutations After mutation at the hgprt locus, the mutant phenotype requires a period of time before it is completely expressed (expression requires the loss of pre-existing enzyme activity). Phenotypic expression is presumably achieved by dilution of the pre-existing H G P R T enzyme and m R N A through cell division and macromolecular turnover. At the normal population doubling times of 12-16 h for CHO cells, an expression period of 7 - 9 days is generally adequate (O'Neill et al., 1977; O'Neill and Hsie, 1979a). The most widely employed method for phenotypic expression allows exponential growth of the cells for a defined time period after mutagen treatment. CHO cells can be subcultured with 0.05% trypsin with or without EDTA. Aliquots of 1 x 106 cells are subcultured at 2- or 3-day intervals in 100-mm diameter tissue culture dishes or 75-cm2 t-flasks. Either complete medium or hypoxanthine-free medium can be employed, with either dialyzed or nondialyzed serum. It is important to ensure that the medium employed will allow a population doubling time of 12-16 h. Besides the normal growth of cells as monolayer cultures, alternative methods of subculturing involving suspension (Carver et al., 1980), unattached (Li, 1981), and division-arrested (O'Neill, 1982) cultures have also been successful. The use of a particular subculture regimen in the expression period should be substantiated by data demonstrating the achievement of optimal expression.
(3) Mutant selection Conditions for the selection of mutants must be defined to ensure that only mutant cells are able to form colonies and that all mutant cells form colonies. In general, cells are plated in tissue culture dishes for attached colony growth (O'Neill et al., 1977; O'Neill et al., 1977), or in agar for suspended colony growth (Li and Shimizu, 1983). An advantage of the former is that after the colonies are fixed and stained, the plates can be counted at a later date. An advantage of the latter is that metabolic cooperation between wild-type and mutant cells is reduced, allowing selection of a higher cell number per plate. Reliable selection has been established in hypoxanthine-free medium containing dialyzed serum
and 10 ~M 6TG. Fetal bovine serum, newborn bovine serum, or calf serum can be used, providing that the serum has been adequately tested and shown to support the desirable characteristics of CHO cells as described above. Dialyzed serum is usually necessary to eliminate the competition between 6TG and purine bases in the serum. It has been found that a selection cell density of 2 x 105 or fewer cells per 100-mm dish for attached colony growth (O'Neill et al., 1977; O'Neill and Bowman, 1982) and 10 6 o r fewer cells per 100-mm dish (in 30 ml agar) for agar colony growth (Li and Shimizu, 1983) allows essentially 100% recovery of mutant cells.
Data presentation Results from the assay should include the following experimental data: (1) Concentrations and solvents used for the test article and positive controls. (2) Absolute and relative cloning efficiencies (CE) in the concurrent cytotoxicity assay, where Absolute CE -
number of coloniesformed number of cells plated
Relative CE =
CE (treatment) CE (solventcontrol)
(3) Actual number of mutant colonies observed for each treatment condition. (4) Absolute CE at selection for each treatment condition. (5) Mutant frequency (MF) values, where MF=
number of mutant colonies number of clonable cells
and Number of colonablecells = number of cells plated × absolute CE at selection MF is usually expressed as mutants per clonable cells.
10 6
Criteria for data acceptability Generally, for the data of a given assay to be acceptable, the following criteria should be met:
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(1) The absolute CE of the negative controls should not be less than 50%. Absolute CE values lower than 50% would indicate suboptimal culturing conditions for the cells. (2) The mean mutant frequency of the solvent controls in each experiment should fall within the range of 0-20 mutants per 1 0 6 clonable cells. A higher mutant frequency may preclude detection of weak mutagens. Under such conditions data acceptability should be evaluated on a case by case basis. (3) The positive control must induce a statistically significant response at a magnitude appropriate for the mutagen under the chosen experimental conditions. (4) The highest test article concentration should, if possible, result in a significant cytotoxic response (e.g. 10-30% survival, where survival is the percent of the treated population that is viable after treatment). This is particularly important if the response is negative. For noncytotoxic test articles, the highest concentration has generally been 1-10 mg per ml, or to the limit of solubility.
Data analysis Due to possibility of fluctuation, only samples with fewer than 105 viable cells after treatment should be used with caution for data analysis. Judgement on mutagenicity should be made based on the following: (1) Dose-reponse relationship. (2) Significance of response (in comparison to the negative control). (3) Reproducibility of the results. Exact statistical analysis is difficult because the distribution of the number of mutant colonies depends on the complex processes of cell growth and death after mutagen treatment. While other appropriate methods can be used, two commonly used approximate methods are as follows: (1) A weighted regression analysis where the weights are proportional to the observed number of mutant colonies divided by the square of the observed mutant frequency (Hsie et al., 1975). This weighting scheme was derived by assuming that the variance of the observed mutant frequency is a constant multiple of that which would occur if the number of mutant colonies on each selection
plate has a Poisson distribution. A test compound is considered to exhibit a mutagenic response if the slope of the mutant induction as a function of test concentrations is greater than zero at the 0.01 level according to the t-test (Tan and Hsie, 1981). (2) A power transformation procedure with which the observed mutant frequency is transformed using the formula Y = ( X + A ) B, w h e r e Y = transformed mutant frequency X = o b s e r v e d m u t a n t f r e q u e n c y , and A, B = c o n s t a n t s
Data transformed by this method appears to satisfy the assumptions of homogeneous variance and normal distribution (Snee and Irr, 1981). Comparison to negative control values and doseresponse relationships are examined with Student's t-test and an analysis of variance (ANOVA) using the transformed values. Computations can be done with computer programs readily available.
Testing strategy In general, the mutagenicity test should b e designed to consider the following: (1) The test substance should be tested at levels allowing significant chemical-cell interaction, which is generally indicated by cytotoxicity: Relatively insoluble chemicals should be tested to the limit of solubility. Nontoxic but highly soluble chemicals should be tested to an arbitrary maximum concentration based on the anticipated human exposure level and a conservative safety factor. As a general rule of thumb, 1-10 m g / m l should be sufficient as the maximum concentration. (2) Different amounts of Aroclor 1254-induced liver $9 may be used, since it has been shown that some mutagens may be highly sensitive to the level of $9 used (Machanoff et al., 1981; Li, 1984). (3) The observation should be reproducible as indicated by two or more independent experiments. (4) In each experiment, intra-experimental variations should be determined using replicate treatment cultures. An example of an adequate combination of experiments (Li, 1985) is as follows: ( a ) Expt. 1. Range-finding for cytotoxicity. Log
140 or half-log concentrations of the test articles are evaluated in the absence and presence of various levels of $9. Cytotoxicity information obtained is used for dose selection in the subsequent m u t a genesis experiments. A repeat of the experiment using a narrower concentration range m a y be necessary for test articles with steep cytotoxic responses.
( b ) Expt. 2. Initial mutagenicity determination with limited doses and at multiple $9 concentrations. This experiment should yield information for an initial estimation of mutagenicity as well as any effects of $9 concentration on mutagenicity. Concentrations of the test article are selected based on the results of Expt. 1.
(c) Expt. 3. Confirmatory mutagenicity determination. This experiment would incorporate the optimal $9 level, if any, as determined in Expt. 2. A larger n u m b e r of concentrations than in Expt. 2 should be used for a more accurate estimation of the d o s e - r e s p o n s e relationship. General guidelines for the performance of this assay for chemical testing have also been published, and can be used as a basis for experimental design (e.g. O'Neill and Hsie, 1979b; Hsie et al., 1981; EPA Health Effects Test Guidelines, 1982; O E C D , 1983). Other considerations Based on the latest scientific information and practical experience, we have developed the above guidelines for the performance of the C H O / H G P R T assay. We c a n n o t overemphasize the fact that these are merely guidelines. This d o c u m e n t should not be viewed as encompassing the only available, appropriate or useful protocols and procedures. There is no substitute for sound scientific judgement and " h a n d s o n " experience. This document, therefore, should not be construed as an instrument for inhibiting present or future research and development towards further refinem e n t of the assay. Being an extensively characterized assay, the CHO/HGPRT assay should be useful in the toxicological evaluation of industrial and environmental substances. A n advantage of using C H O cells is that other well-characterized genotoxicity endpoints have also been developed, e.g., muta-
tions at other gene loci (Carver et al., 1980; G u p t a and Siminovitch, 1980; G u p t a and Singh, 1982; Singh and Gupta, 1983a; Sing and Gupta, 1983b), c h r o m o s o m a l aberrations (Preston et al., 1981), and sister-chromatid-exchanges (Wolff, 1977). It is therefore possible to use a variety of endpoints in C H O cells for testing, yielding additional information that m a y be used, in conjunction with data from other toxicity assays, for the prediction of the h u m a n toxicological consequences of exposure to the substances tested. Acknowledgement The authors are grateful for the effort of Dr. Robert Naismith who, as a chariman of the A S T M subcommittee on Genetic Toxicology, has been instrumental for the initiation of the C H O / H G P R T work group. References Carver, J.H., G.M. Adair, and D.L. Wandres (1980) Mutagenicity testing in mammalian cells, II. Validation of multiple drug resistance markers having practical application for screening potential mutagens, Mutation Res., 72, 207-230. EPA Health Effects Test Guidelines (1982) EPA 560/6-82-001. Gupta, R.S., and L. Siminovitch (1980) Genetic markers for quantitative mutagenesis studies in Chinese hamster ovary ceils: Characteristics of some recently developed selective systems, Mutation Res., 69, 113-126. Gupta, R.S., and B. Singh (1982) Mutagenic response in five independent genetic locus in CHO cells to a variety of mutagens: Development and characteristics of a mutagen screening system based on selection for multiple drug resistant markers, Mutation Res., 94, 449-466. Hsie, A.W., P.A. Brimer, T.J. Mitchell and D.G. Gosslee (1975) The dose-response relationships for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells, Somatic Cell Genet., 1, 247-261. Hsie, A.W., A.P. Li and R. Machanoff (1977) A fluence-response study of lethality and mutagenicity of white, black, and blue florescent light, sunlamp, and sunlight irradiation in Chinse hamster ovary cells, Mutation Res., 45, 333-342. Hsie, A.W., J.P. O'Neill, D.B. Couch, J.R. SanSebastian, P.A. Brimer, R. Machanoff, J.C. Riddle, A.P. Li, J.C. Fuscoe, N.L. Forbes and M.H. Hsie (1979) Utilization of a quantitative mammalian cell mutation system in experimental mutagenesis and genetic toxicology, in: B. Butterworth (Ed.), Strategy for Short-Term Testing for Mutagens/ Carcinogens, C.R.C. Press, West Palm Beach, pp. 39-54. Hsie, A.W., D.A. Casiano, D.B. Couch, D.F. Krahn, J.P. O'Neill and B.L. Whitfield (1981) The use of Chinese hamster ovary cells to quantify specific locus mutation and
141 to determine mutagenicity of chemicals, A report of the Gene-Tox program, Mutation Res., 86, 193-224. Li, A.P. (1981) Simplification of the C H O / H G P R T mutation assay through the growth of Chinese hamster ovary cells as unattached cultures, Mutation Res., 85, 165-175. Li, A.P. (1984) Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells, Environ. Mutagen., 6, 539-544. Li, A.P. (1985) A testing strategy to evaluate the mutagenic activity of industrial chemicals in cultured mammalian cells, Regul. Toxicol. Pharmacol., 5, 207-211. Li, A.P., and R.W. Shimizu (1983) A modified agar assay for the quantitation of mutation at the hypoxanthine guanine phosphoribosyl transferase gene locus in Chinese hamster ovary cells, Mutation Res., 111, 365-370. Li, A.P., A.L. Brooks, C.R. Clark, R.W. Shimizu, R.L Hansen and J.S. Dutcher (1983) Mutagenicity testing of complex environmental mixtures with Chinese hamster ovary cells, in: M. Waters, S. Sandbu, J. Lewtas, L. Claxton, M. Chernoff and S. Nesnow (Eds.), Short-term Bioassays in the Analysis of Complex Environmental Mixtures III, Plenum, New York, pp. 183-196. Machanoff, R., J.P. O'Neill and A.W. Hsie (1981) Quantitative analysis of cytotoxicity and mutagenicity of benzo(a)pyrene in mammalian cells ( C H O / H G P R T system), Chem.-Biol. Interact., 34, 1-10. OECD (1983) Guidelines for Testing of Chemicals: Genetic Toxicology. In vitro Mammalian Cell Gene Mutation Tests (final draft). O'Neill, J.P. (1982) Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division, Mutation Res., 106, 113-122. O'Neill, J.P., and K.O. Bowman (1982) Effect of selection cell density on the recovery of mutagen induced TG r cells, Environ. Mutagen., 4, 627-637. O'Neill, J.P., and A.W. Hsie (1979a) Phenotypic expression time of mutagen induced 6-thioguanine resistance in CHO ceils, Mutation Res., 59, 109-118. O'Neill, J.P., and A.W. Hsie (1979b) C H O / H G P R T Mutation
Assay: Experimental Procedure, in: A.W. Hsie, J.P. O'Neill and V.K. McElheny (Eds.), Banbury Report 2, Mammalian Cell Mutagenesis: The Maturation of Test Systems, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 55-69. O'Neill, J.P., P.A. Brirner, R. Machanoff, G.P. Hirsch and A.W. Hsie (1977) A quantitative assay of mutation induction at the hgprt locus in CHO cells, Mutation Res., 45, 91-101. Preston, R.J., W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McFee, S. Wolff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenesis assay: A report of the U.S. Gene-Tox program, Mutation Res., 87, 143-188. Riddle, J.C., and A.W. Hsie (1978) An effect of cell cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells, Mutation Res., 52, 409-420. Singh, B., and R.S. Gupta (1983a) Comparison of the mutagenic responses of twelve anticancer drugs at the hypoxanthine-guanine phosphoribosyl transferase and adenosine kinase loci in Chinese hamster ovary cells, Environ. Mutagen., 5, 871-880. Singh, B., and R.S. Gupta (1983b) Mutagenic responses of thirteen anticancer drugs on mutation induction at multiple genetic loci and on sister chromatid exchanges in Chinese hamster ovary cells, Cancer Res., 43, 577-584. Snee, R.D., and J.D. Irr (1981) Design of a statistical method for the analysis of mutagenesis at the hypoxanthine-gnanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells, Mutation Res., 85, 77-93. Tan, E.-L., and A.W. Hsie (1981) Mutagenicity and cytotoxicity of haloethanes as studied in the C H O / H G P R T system, Mutation Res., 90, 183-191. Wolff, S. (1977) Sister chromatid exchange, Annu. Rev. Genet., 11, 183-201. Yuhas, J.M., A.P. Li and M.N. Kligerman (1979) Present status of the proposed of negative pimesons in radiotherapy, in: J.T. Lett and H. Adler (Eds.), Advances in Radiation Biology, Vol. 8, Academic Press, New York, pp. 51-85.