Enumeration of thioguanine-resistant lymphocytes using autoradiography

doi:10.1016/0027-5107(82)90271-8

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 95, Issues 2–3, August 1982, Pages 363-375

https://doi.org/10.1016/0027-5107(82)90271-8 Get rights and content

Abstract

Strauss and Albertini (1979) proposed a method whereby the frequency of thioguanine-resistant lymphocytes could be measured by an autoradiographic technique and suggested that it would provide a measure of in vivo mutation frequency. A number of variables influencing the method were studied and concentration of thioguanine and duration of assay found to be particularly important. The modified method finally arrived at involved culture of PHA-stimulated lymphocytes for 64 h in the presence of 40 μg/ml of thioguanine, lysis of cells with Nonidet and collection of nuclei on Nucleopore membranes. The frequency of TG-resistant cells was 1.5×10⁻⁶−1.7×10⁻⁵ in young adults and it showed an increase with age. The technique was also adapted for use with continously cultured lymphocytes, by using a concentration of 60 μg/ml of thioguanine and an incubation time of 72 h. The frequency of TG-resistant continously cultured cells was <10⁻⁶−2×10⁻⁵.

Consideration of the effect of different variables on the assay suggested that the above estimates for TG-resistant lymphocytes were more valid than the previously published substantially higher estimates.

References (21)

C.T. Caskey et al.
The HGPRT locus
Cell
(1979)
R. Cox et al.
X-ray-induced mutation to 6-thioguanine resistance in cultured human fibroblasts
Mutation Res.
(1976)
R.S. Gupta et al.
Genetic markers for quantitative mutagenesus studies in Chinese hamster ovary cells: Characteristics of some recently developed systems
Mutation Res.
(1980)
G.H. Strauss et al.
Enumeration of 6-thioguanine-resistant peripheral blood lymphocytes in man as a potential test for somatic cell mutations arising in vivo
Mutation Res.
(1979)
J. Thacker et al.
Factors affecting the frequency of purine analogues as selective agents for mutants of mammalian cells induced by ionizing radiation
Mutation Res.
(1976)
W.O. Thilly et al.
Phenotypic lag and mutation to 6-thioguanine resistance in diploid human lymphoblasts
Mutation Res.
(1978)
A.A. Van Zeeland et al.
The effect of calf serum on the toxicity of 8-azaguanine
Mutation Res.
(1975)
A.A. Van Zeeland et al.
The use of correction factors in the determination of mutant frequencies in populations of human diploid skin fibroblasts
Mutation Res.
(1976)
R.A. Walters et al.
Effects of ionizing radiation on nucleic acid synthesis in mammalian cells
Adv. Radiation Biol.
(1976)
R.J. Albertini
Direct mutagenicity testing with peripheral blood lymphocytes

There are more references available in the full text version of this article.

Cited by (30)

Strategies and testing methods for identifying mutagenic risks
2000, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Citation Excerpt :
The most developed and validated endogenous in vivo system measures mutations at the hprt locus in mammalian lymphocytes. The development of the hprt assay employing human peripheral blood lymphocytes has contributed significantly to our understanding of in vivo somatic cell mutagenesis in the human [92–97]. In vivo chemical/physical mutagenesis assays at the hprt locus in lymphocytes of rodents and non-human primates have also been developed in lymphocytes [98–101], such that the same endpoint can be monitored in laboratory animals or in epidemiology studies involving human subjects.
The evolution of testing strategies and methods for identification of mutagenic agents is discussed, beginning with the concern over potential health and population effects of chemical mutagens in the late 1940s that led to the development of regulatory guidelines for mutagenicity testing in the 1970s and 1980s. Efforts to achieve international harmonization of mutagenicity testing guidelines are summarized, and current issues and needs in the field are discussed, including the need for quantitative methods of mutagenic risk assessment, dose–response thresholds, indirect mechanisms of mutagenicity, and the predictivity of mutagenicity assays for carcinogenicity in vivo. Speculation is offered about the future of mutagenicity testing, including possible near-term changes in standard test batteries and the longer-term roles of expression profiling of damage-response genes, in vivo mutagenicity testing methods, and models that better account for differences in metabolism between humans and laboratory model systems.
Bromodeoxyuridine labelling as an alternative method to identify 6- thioguanine-resistant mutant lymphocytes in humans
1999, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
6-Thioguanine-resistant (TG^R) mutant lymphocytes in human blood are usually enumerated by the cloning assay which allows the molecular characterisation of the HPRT mutations to be detected. A “short-term” alternative approach is provided by the anti-bromodeoxyuridine (anti-BrdU) technique in which TG^R lymphocytes are identified immunocytochemically by their ability to synthesise DNA in the presence of 6-thioguanine (TG). We have evaluated the influence of various experimental factors that could affect the frequency of TG^R lymphocytes. A standard protocol is proposed, based on 24-h cold storage of isolated lymphocytes at 4°C and 40-h culture with and without TG, the last 16 h with BrdU. The harvested cells are treated with hypotonic (0.075 M) KCl, fixed with methanol:acetic acid (3:1) and put on microscopic slides. For the TG cultures, all cells are prepared on the slides, while slides from the control cultures are made by a 1/50 dilution. DNA is denatured by formamide, and the BrdU label is identified by anti-BrdU antibody detected by immunoperoxidase staining using a peroxidase-conjugated secondary antibody with diaminobenzidine as substrate. In 10 donors, the frequency of TG^R lymphocytes (variant frequency, V_f) detected by this protocol ranged from 69.65×10⁻⁶ to 83.45×10⁻⁶, and split measurements showed a relatively small intra-assay variation in V_f values of each donor. BrdU in DNA was also detected by immunofluorescence using a fluorescein-conjugated anti-BrdU monoclonal antibody. This method, facilitating easy identification of positive cells and rapid microscopic scoring, may serve as a basis for an automated analysis of TG^R lymphocytes. V_f values detected by the anti-BrdU assay are higher than mutant frequencies obtained by the cloning assay, which has been assigned to the presence of non-mutant phenocopies considered to represent spontaneously cycling lymphocytes. Although the anti-BrdU assay is rapid and easy and has been shown to respond to genotoxic exposures, its true value could be evaluated only when it can be ascertained that phenocopies do not significantly contribute to the V_f values obtained.
Development and utilization of the rat lymphocyte hprt mutation assay
1997, Mutation Research - Reviews in Mutation Research
Much of the recent progress in the field of genetic toxicology has come from an increased understanding of the molecular and cellular biology of the mammalian organism. Most prominent has been the ability to detect and quantify somatic mutation and relate the nature of the mutation to the specific type of chemical damage. Building upon the foundation of the human lymphocyte hypoxanthine guanine phosphoribosyl transferase (hprt) system, and later, the mouse hprt system, methods for the detection and quantification of hprt mutations in rat lymphocytes were developed. These methods are described in this report as is the ongoing validation of the assay. Additionally, the characterization of the recovered mutants and a comparison of the mutation spectrum in the rat lymphocyte system to the spectrum in cancer genes, such as H-ras and p53, and the spectrum in transgenic systems, such as lacI, are included. The development of the rat lymphocyte hprt system and validation of the assay at the molecular level, provide an effective and reliable measure of genetic damage in an in vivo system which is readily comparable to measurement of genetic damage in the human.
Working paper no. 3 Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo
1994, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations
1993, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Somatic cell mutant frequencies at the hprt locus of the X-chromosome were measured with the T-lymphocyte cloning technique in healthy human populations. A statistical analysis was performed of assays from 232 individuals (77 males and 155 females) ranging in age from 19 to 80 years. Data from 4 donor groups were compiled: (a) 132 participants in a study of identical and fraternal twins; (b) 17 health care workers studied as part of an assessment of the risks of handling chemotherapeutic drugs; (c) 62 women with benign breast masses; and (d) 21 normal laboratory and office personnel. The relationship between age and mutant frequency (MF) was expressed by the equation: In MF = 1.46 + 0.018 age (P<0.001). Thus, MF increased by about 2% per year. Increases in cloning efficiency (CE) reduced the MF, as shown in the equation: In MF = 2.91-1.32 CE (P<0.001). CE was significantly related to age (CE = 0.47-0.002 age, P = 0.038), and the interdependent relationship between MF, age and CE expressed by the equation: In MF = 1.99-1.13 CE + 0.016 age was significant at the P<0.001 level. There was no statistically significant effect of donor gender or smoking history on MF in our population, but CE was significantly lower in males (P< 0.001). These findings confirm the importance of age and CE as factors which influence the thioguanine-resistant MF in circulating T-lymphocytes from normal adults.
Ionizing radiation and genetic risks III. Nature of spontaneous and radiation-induced mutations in mammalian in vitro systems and mechanisms of induction of mutations by radiation
1991, Mutation Research/Reviews in Genetic Toxicology
This paper (1) presents an analysis of published data on the molecular nature of spontaneously arising and radiation-induced mutations in mammalian somatic cell systems and (2) examines whether the molecular nature and mechanisms of origin of radiation-induced mutations, in mammalian in vivo and in vitro systems, as currently understood, are consistent with expectations based on the biophysical and microdosimetric properties of ionizing radiation.
Depending on the test system (CHO cells, human T lymphocytes and human lymphoid cell line TK6), 80–97% of spontaneous HPRT mutations show normal Southern patterns; the remainder is due to gross changes, predominantly partial (intragenic) deletions. Total gene deletions at the HPRT locus are rare except in the TK6 cell line.
At the APRT locus in CHO cells, 80–97% of spontaneous mutations are due to base-pair changes, the remainder being, mostly, partial delections. The latter can extend upstream in the 5′ direction but not beyond the APRT gene in the 3′ direction. At the human HLA-A locus (T lymphocytes), the percentage of mutations with normal Southern patterns is lower than that for HPRT, and in the range of 50–60%. At the HLA-A locus, mitotic recombination contributes substantially to the mutation spectrum (∼ 30% of mutations recovered) and this is likely to be true of the TK locus in the TK6 cell line as well.
With a few exceptions, most of the radiation-induced mutations show altered Southern patterns and are consistent with their being deletions and/or other gross changes (HPRT, 70–90% (CHO); 50–85% (TK6); 50–75% (T lymphocytes); TK, 60–80% (TK6); HLA-A, 80% (T lymphocytes); DHFR, 100% (CHO)). The exceptions are APRT mutations in CHO cells (16–20% of mutants with deletions or other changes) and HPRT mutations in T lymphocytes from A-bomb survivors (15–25%); the latter finding is consistent with the occurrence of in vivo selection against HPRT mutant cells. In cases of HPRT intragenic deletions analyzed (CHO cells and V79 Chinese hamster cells), there is evidence for a non-random distribution of breakpoints.
The spontaneous mutation frequencies vary widely, from about 0.04/10⁶ cells (sickle cell mutations at the human HBB locus) to 30.8/10⁶ cells HLA-A mutations in T lymphocytes) and are dependent on the locus, the system employed and a number of other factors. Those for the other loci fall between these limits.
The rates of induction by X-rays again are quite low for the HBB locus (0.07–1.5/10⁶ cells/Gy; only two types of specific point mutations were scored), low for DHFR (1.6/10⁶ cells/Gy) and APRT (4.3/10⁶ cells/Gy), in the range of about 10–30/10⁶ cells/Gy for most others, and are highest for the HLA-A, and glycophorin A locus (the NØ variant) (about 60/10⁶/cells/Gy). Gene size alone cannot explain the differences between either spontaneous frequencies or induction rates.
On the basis of chromatin and DNA organization in cells and the biophysical and microdosimetric properties of ionizing radiation, one can qualitatively explain the predominance of deletions among radiation-induced mutations. For spontaneously arising and radiation-induced point mutations, there may be common elements in mechanisms, but for spontaneously arising and induced deletions, the extent of overlap in mechanisms is difficult to discern at present.

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Enumeration of thioguanine-resistant lymphocytes using autoradiography

Abstract

Cell

Mutation Res.

Mutation Res.

Mutation Res.

Mutation Res.

Mutation Res.

Mutation Res.

Mutation Res.

Adv. Radiation Biol.

Direct mutagenicity testing with peripheral blood lymphocytes