Mutagenicity monitoring in humans by autoradiographic assay for mutant T lymphocytes

Somatic cell mutation which occurs in vivo in humans can be determined by measurement of the frequency of the 6-thioguanine-resistant (TGr) T lymphocytes in samples of peripheral blood. This frequency can be determined by either a short-term autoradiographic methodology or a longer cell-cloning methodology. The advantage of the former is the relative simplicity of the assay, while the latter allows recovery of mutant clones for further characterization. This report presents results of a longitudinal study of cancer chemotherapy nurses and other health care personnel by use of the autoradiography assay. The use of this assay in human mutagenicity monitoring and the analysis of the TGr cell frequencies are discussed in terms of age effects and validation of 'elevated' frequencies by use of the clonal assay. This report then presents evidence that both assays yield similar TGr cell frequencies in two groups of 'normal adults'. The mean variant frequency (+/- S.D.) for 82 autoradiographic assays was 8.7 (+/- 6.1) X 10(-6), while the mean mutant frequency (+/- S.D.) for 115 clonal assays was 6.5 (+/- 4.8) X 10(-6). In addition, concurrent autoradiographic and clonal assays on 33 individuals yielded mean values (+/- S.D.) of 8.4 (+/- 8.5) X 10(-6) and 10.5 (+/- 6.3) X 10(-6), respectively.     

Direct and indirect flow cytometric enumeration of 6-thioguanine-resistant human peripheral blood lymphocytes

Methods based on flow cytometry and sorting, autoradiography, and cloning were used to evaluate the potential for the enumeration of 6-thioguanine-resistant human peripheral blood lymphocytes assumed to be deficient with respect to the enzyme hypoxanthine-guanine-phosphoribosyl-transferase. Flow cytometric sorting of proliferating cells in the late S- and the G2-stages by means of DNA content, as measured by propidium iodide fluorescence, enabled an enrichment of variant cells to about 99%. The main source of false events was contaminating doublets of G0/G1 cells appearing in the sorting region. Doublet discrimination measured as the difference between pulse height and area (Ortho-50) accomplished no further improvement. A combination of propidium iodide fluorescence and bromodeoxyuridine incorporation, measured by fluorescent anti-bromodeoxyuridine-DNA antibodies, allowed flow cytometric enrichment to about 99.99% of variant cells. By sorting of 3H-thymidine-labeled cell nuclei from the late S- and the G2-phases and subsequent autoradiographic evaluation, partly resistant variants could be discriminated; variant frequencies of the same magnitude as for the cell cloning methods were obtained.     

Somatic gene mutations in vivo as indicated by the 6-thioguanine-resistant T-lymphocytes in human blood

The clonal and the autoradiographic assays for 6-thioguanine-resistant (TG^r) T-lymphocytes (T-Lys) in human blood are reviewed. Studies of TG^r colonies recovered from clonal assays show that the mutant T-Lys (i) are either helper (T4) or suppressor (T8) cells, (ii) poses stable TG^r phenotype, (iii) are deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT), and (iv) have structural alterations in the hprt gene. TG^r T-Ly mutant frequencies (Mfs) determined by clonal assays are of the order of 10⁻⁶−10⁻⁵ for normal adults. Autoradiographically determined variant frequencies (Vfs) are also in this range for normal adults when lymphocytes are cryopreserved before study to remove ‘phenocopies’. Cancer exposed to potentially mutagenic treatments have elevated TG^r T-Ly Vfs. Comparative clonal and autoradiographic assays of the same blood samples give generally similar results when allowances are made for potential sources of error in each assay. The TG^r T-Ly system is presented for human specific-locus mutagenicity monitoring.     

6-Thioguanine resistant T-lymphocyte determination as a possible indicator of human ionizing radiation exposure

We used the autoradiographic assay to assess human in vivo somatic cell gene mutation at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in T-lymphocytes. Cells able to incorporate tritiated thymidine in vitro in 6-thioguanine containing short-term cultures were enumerated in order to determine 6-thioguanine resistant (TGr) variant frequencies in cryopreserved lymphocytes from control individuals and 3 persons suspected of 60Co exposure from an accident in Cd. Juárez, México. The data indicate that the lymphocyte TGr variant frequency assay may be potentially usefull for human population monitoring following accidental exposures to ionizing radiation.     

Elevated frequencies of 6-thioguanine-resistant lymphocytes in multiple sclerosis patients treated with cyclophosphamide: a prospective study

An autoradiographic assay for 6-thioguanine-resistant (TGr) lymphocytes was used to determine the frequency of in vivo derived variant T lymphocytes in peripheral blood from multiple sclerosis (MS) patients treated with monthly intravenous infusions of 750 mg/m2 of cyclophosphamide (CP). To analyze the time-course of response to CP, the MS patients were studied prospectively. Samples were obtained from the patients before the beginning of CP therapy, 4-5 times during the course of treatment, and, finally, 2 or 3 months after the completion of therapy. 2 weeks after the first CP infusion, the variant frequencies (Vfs) of the MS patients were significantly increased (p less than 0.05) above their pre-treatment values, but by 4 weeks following the first CP infusion the Vfs had fallen to normal or near-normal levels. After subsequent treatments, the frequencies of variant TGr cells were again higher than pre-treatment Vfs. However, within 7-13 weeks after the cessation of CP therapy, the Vfs of all subjects had returned to normal levels. The transient nature of the response indicates rapid in vivo selection against CP-induced TGr mutant cells. The mean pre-treatment Vf of the 4 MS patients who were cigarette smokers was 6.56 X 10(-6) which was significantly greater (p less than 0.05) than the mean Vf (1.52 X 10(-6) of the 4 MS patients who were non-smokers. The mean Vf from 8 assays of healthy non-smokers was 1.92 X 10(-6).     

Autoradiographic detection of HPRT variants of human lymphocytes resistant to RNA synthesis inhibition

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.     

Autoradiographic detection of 6-thioguanine-resistant lymphocytes of mice: A novel system in somatic mutagenesis testing

After treatment of mice with ethylnitrosourea in vivo, induction of 6-thioguanine-resistant splenic lymphocytes was measured in vitro. The lymphocytes were mitogen-stimulated in presence of thioguanine. After a culture period of 30 h, TG resistance was determined by the ability of cells to incorporate [³H]thymidine. The results suggest that at this selection condition the majority of resistant cells stem from somatic mutations. Thus, the system is potentially useful as a screening assay for somatic mutagenesis testing. The problem of misleading results due to the presence of phenocopies was investigated by treatment in vitro with UV radiation or ethylnitrosourea.     

International Commission for the Protection of the Environment against Mutagens and Carcinogens: a historical perspective

Hypoxanthine guanine phosphoribosyl transferase (HPRT) deficient human peripheral blood lymphocytes are usually enumerated either by the cloning assay or by the autoradiographic short-term assay. The short-term approach presented here is based on flow cytometric (FCM) scoring of 6-thioguanine (6-TG) resistant lymphocytes. HPRT-variants are enumerated on the basis of both DNA synthesis (by use of immunofluorescent detection of incorporated 5-bromo-2-deoxyuridine, BrdU) and total DNA content (by propidium iodide (PI) incorporation) of proliferating cells, i.e. the cells must both be labelled with BrdU and reside in late-S or G2 phase in order to be scored as a HPRT-variant. This approach is combined with a stringent discrimination of false-positive events, minimising occurrence of phenocopies or other non-specifically labelled cells that might falsely be scored as true HPRT-variants. The HPRT-variant frequency (V(f)) found by the presented method varied between 0.8 x 10(-5) and 5.8 x 10(-5) for healthy male and female donors aged between 20 and 74 years. There was no significant gender difference in V(f). A strong linear correlation was found between HPRT-variant frequency and age, showing an increase of 0.56 x 10(-6) per year of age (r(2)=0.62, P<0.001). The frequencies of false-positive events found showed a mean of 0.22 x 10(-5) in comparison with a pooled mean V(f) of 2.87 x 10(-5). There was no significant age effect on the frequency of false events (r(2)=0.15, P<0.095). The method presented here may provide a rapid and sensitive alternative to the autoradiographic technique for the short-term enumeration of HPRT-variants.     

HO-323, a human B-lymphoblastoid cell line useful for making human-human hybridomas

A 6-thioguanine-resistant human B-lymphoblastoid cell line, HO-323, was isolated for making human-human hybridomas with high efficiency. Fusions with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients and lymphocytes isolated from lymph nodes of lung and breast cancer patients yield constantly more than one hybridoma clones per 10(5) HO-323 cells plated. HO323 cells also fused with lymphocytes from normal peripheral blood to give hybridomas in the same efficiency. The HO-323 cells were diploid with 46 chromosomes and non-secretors of immunoglobulins. This parent cells doubled every 15 hr and could proliferate in serum-containing medium, even if they were plated at low cell density of less than 10(3) cells/ml. The cells could grow in serum-free medium as well as in serum-containing medium, and the resultant human-human hybridomas could also grow in the same media.     

A quantitative assay for measuring the induction of mutations in human peripheral blood T-lymphocytes

We optimized conditions for propagating freshly-isolated human peripheral blood T-lymphocytes and cells that had been stored in liquid nitrogen on Day 5 post-isolation, exposing them to mutagens in exponential growth, and measuring the cytotoxicity of the agent from the loss of colony-forming ability, and its mutagenicity from the increase in frequency of 6-thioguanine-resistant cells. Supernatant containing T-cell growth factor, from 60Co-irradiated peripheral mononuclear cells cultured in the presence of 60Co-irradiated B-lymphoblastoid human cells as allogeneic stimulators, supplied at a concentration of 10% along with 10% serum and 10(5) allogeneic stimulator cells/ml, supported exponential growth (population doubling times of 22 h) for extended periods (greater than 30 d). It gave cloning efficiencies of greater than or equal to 40%. T-lymphocytes stored in liquid nitrogen and returned to culture shortly before mutagen exposure exhibited the same sensitivity as freshly-isolated T-cells to killing by the agents tested, i.e., UV radiation, ethyl-nitrosourea, and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,19 alpha,epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene. We showed that if mutagenized populations frozen during the expression period were thawed and assayed, they exhibited the same cloning efficiencies and frequencies of 6-thioguanine-resistant cells as did the corresponding populations that had been assayed directly without freezing. Use of these procedures should facilitate investigation of the frequency and kinds of mutations induced in the HPRT gene of peripheral blood T-lymphocytes in vivo and in vitro.     

Detection and long-term in vivo monitoring of individual tumor-specific T cell clones in patients with metastatic melanoma

We investigated the presence of individual melanoma-specific T cell clones in patients with metastatic melanoma. Ten patients were examined for the presence of melanoma-reactive T cells using dendritic cells loaded with autologous tumor cells. Their specificity was tested using nonradioactive cytotoxicity test. Individual immunodominant T cell clones were identified by the clonotypic assay that combines in vitro cell culture, immunomagnetic sorting of activated IFN-gamma(+) T cells, TCRbeta locus-anchored RT-PCR, and clonotypic quantitative PCR. All patients had detectable melanoma-reactive T cells in vitro. Expanded melanoma-reactive T cells demonstrated specific cytotoxic effect against autologous tumor cells in vitro. Three patients experienced objective responses, and their clinical responses were closely associated with the in vivo expansion and long-term persistence of individual CD8(+) T cell clones with frequencies of 10(-6) to 10(-3) of all circulating CD8(+) T cells. Five patients with progressive disease experienced no or temporary presence of circulating melanoma-reactive T cell clones. Thus, circulating immunodominant CD8(+) T cell clones closely correlate with clinical outcome in patients with metastatic melanoma.     

Activation through CD3 molecule leads to clonal expansion of all human peripheral blood T lymphocytes: functional analysis of clonally expanded cells

Monoclonal antibodies directed against CD3, a T cell-specific surface molecule essentially required for activation of these cells, are highly mitogenic for resting human peripheral blood T lymphocytes. A predetermined optimal concentration of anti-CD3 monoclonal antibody WT32 was employed to activate T cells cultured in limiting-dilution microcultures containing irradiated feeder cells and exogeneous interleukin 2. Frequencies of cells triggered into clonal expansion by WT32 under these culture conditions were 0.57 to 0.72 and 0.90 to 1.10 in peripheral blood mononuclear cells and E rosette-positive cells, respectively. It appeared that WT32 could induce virtually every human peripheral blood T lymphocyte to expand into a clonal progeny of 5 to 40 X 10(4) cells in 14 to 18 days of culture. This progeny was tested for cytolytic effector function with 51Cr-labeled murine P815 targets in the presence of PHA to detect all cytotoxic T lymphocytes (CTL) regardless of specificity, and was also assayed for natural killer like activity against K562 target cells. Frequencies of cells in the human peripheral blood T cell compartment giving rise to a clonal progeny expressing CTL function was 1/3, whereas 1/6 to 1/5 expanded into effector cell populations possessing NK activity. Frequency analysis of CD4-positive and CD8-positive populations, activated by WT32 in limiting dilution microcultures, demonstrated that 1 to 6% of the CD4-positive and 100% of the CD8-positive peripheral blood T lymphocytes expanded into CTL.     

Immunological studies of aging. IX. Impaired proliferation of T lymphocytes detected in elderly humans by flow cytometry

Lymphocytes from humans over the age of 65 incorporate approximately 50% less tritiated thymidine than do lymphocytes from young donors when cultured with phytohemagglutinin. Because lymphocytes from elderly humans are more sensitive to cell cycle arrest induced by tritiated thymidine, it was impossible to determine to what extent impaired thymidine incorporation reflected a defect in proliferation or the increased sensitivity to the radioactive isotope. Flow cytometry was used to measure the proliferative response of T cells from young and old donors in culture with PHA. It was found that 25 percent fewer lymphocytes from old as compared to young humans enter the G1 or complete the S phase of the cell cycle. However, the rate of progression through the cell cycle by activated cells from young and old humans is comparable. Thus, flow cytometry suggested that the difference in thymidine incorporation by lymphocytes from old and young donors is attributable equally to a proliferative defect and to cell cycle arrest induced by tritiated thymidine. This conclusion was supported by the fact that the relative impairment of thymidine incorporation by lymphocytes from old donors was only one-half as great when a 20-min instead of a 24-hr pulse of tritiated thymidine was used.     

Regulation of interleukin 2 receptor expression: effects of phorbol diester, phospholipase C, and reexposure to lectin or antigen

Anti-Tac monoclonal antibody identifies the receptor for interleukin 2 (IL 2, or T cell growth factor) present on activated human T lymphocytes. By using tritiated anti-Tac, we now report a sensitive and specific binding assay to evaluate cell surface IL 2 receptor expression. IL 2 receptors on human peripheral blood lymphocytes can be detected within 6 hr after PHA stimulation. PHA-induced receptor expression is inhibited by actinomycin D and cycloheximide, but not by mitomycin C, suggesting a requirement for de novo RNA and protein synthesis, but not DNA synthesis. Scatchard analysis of [3H]-anti-Tac binding to lymphocytes stimulated with PHA for 3 days revealed from 20,000 to 60,000 molecules of antibody bound per cell, and a Kd of 1 to 3 x 10(-10) mol/l. Sequential binding studies of activated human lymphocytes maintained in long-term culture with IL 2 demonstrated a progressive decline in receptor number correlating with diminished growth rate. Restimulation with lectin or antigen increased the number of IL 2 receptors, suggesting that IL 2 dependent immune responses may be regulated, at least in part, by IL 2 receptor expression. Receptor number was also increased by PMA. Moreover, similar effects were produced by incubation with phospholipase C but not interleukin 1. Because both PMA and phospholipase C result in activation of protein kinase C, these data suggest the possibility that activation of protein kinase C may induce IL 2 receptor expression.     

Analysis of oxidative DNA damage and HPRT mutant frequencies in cancer patients before and after radiotherapy

Various markers of radiation-induced DNA damage including DNA oxidation were investigated in peripheral lymphocytes of 23 cancer patients prior to and one week after receiving radiotherapy with a cumulative dose of 54-70 Gy. Exposure to ionizing radiation nonsignificantly increased the ratio 2'deoxy-7-dihydro-8-oxoguanosine/2'deoxyguanosine (8-oxodG/dG) from 1.73 x 10(-5) to 3.33 x 10(-5). Frequencies of micronuclei significantly (p = 0.0003) increased from 6.4 to 38.9 per 1000 cells. The frequency of hypoxanthine-guanine-phosphoribosyltransferase (HPRT) mutant lymphocytes measured as 6-thioguanine resistant variant cells by 5-bromodeoxyuridine labeling, was elevated eight-fold, from 4.7 x 10(-6) to 36.2 x 10(-6) (p = 0.008) after termination of the radiotherapy, thus showing a clear response to the radiation treatment. No correlation between levels of oxidative DNA damage and frequencies of HPRT mutant lymphocytes or micronuclei could be established.     

Cytotoxic and mutagenic effects of emodin on cultured mouse carcinoma FM3A cells

Employing a suspension culture of a mouse mammary carcinoma cell line, FM3A cells, the cytotoxicity and induced mutagenicity of emodin (EM) were examined and compared with those of 2-hydroxy-emodin (2-OH-EM), which was identified as an active form of EM in the Ames/microsomes assay. EM was cytotoxic to FM3A cells in concentrations of 1-10 micrograms/ml, and induced 6-thioguanine-resistant (6TGr) mutation. 2-OH-EM was a little more toxic than EM, but induced little mutation.     

Attempts to use the HPRT-assay as an automated short-term monitor for an acute exposure to mutagens

Attempts have been made to use the hypoxanthine-guanine-phospho-ribosyl-transferase-assay as a method for automated screening of agent-induced phenotypic variants of human peripheral lymphocytes reflecting 6-thioguanine resistance and assumed to indicate genotoxic action. Different protocols of the hypoxanthine-guanine-phospho-ribosyl-transferase-system were used in this study in order to investigate whether the system can be a candidate for a short-term test for a rapid and reliable identification of biological systems exposed to agents. The current protocols were based on: 1) fluoresceinated monoclonal antibodies against bromodeoxyuridine-DNA for labelling of 6-thioguanine-resistant human lymphocytes and direct flow-cytometric enumeration of bromodeoxyuridine-positive events and: 2) indirect flow-cytometric enrichment of 6-thioguanine-resistant cells labelled with ³H-thymidine followed by autoradiographic enumeration of positive events. Both the direct and the indirect enumeration method yielded similar results down to the range 10^–4 with respect to frequency of variants. For the less time-consuming direct enumeration method the resolution was limited due to non-specific binding of the antibody and false positives. It was, nevertheless, sufficient to score variants induced in vitro with the mutagens EMS, MMC and TT in the same range as e.g. that of cancer patients during and after chemotherapy or radiotherapy, or that of psoriasis patients during and after PUVA (8-methoxypsoralen and long range UV light)-therapy. We conclude that the direct enumeration protocol can be used for a rapid screening of so called outliers, but a more sensitive test, such as the more time-consuming enrichment protocol based on autoradiography, must be used in order to score variants in the range 10^–5–10^–6 Abbreviations ³H-TdR Tritiated thymidine - BrdU 5-2-bromodeoxyuridine - DNA Deoxyribonucleic acid - EMS Ethyl-methanesulphonate - FBS Fetal bovine serum - FITC Fluorescein Isothiocyanate - G₀ Cell cycle stage - G₁ Cell cycle stage - G₂ Cell cycle stage - HPRT Hypoxanthine Guanine Phospho Ribosyl Transferase - LI Labelling index - MMC Mitomycin C - PBL Peripheral blood lymphocytes - PBS Phosphate buffered saline - PHA Phytohemagglutinin - PI Propidium iodide - PUVA 8-Methoxypsoralen and long range UV-light therapy - RNA Ribonucleic acid - RPMI RPMI-1640 culturing medium - S_e Early S-stage in the cell-cycle - S_l Late S-stage in the cell-cycle - SCE Sister chromatid exchange - TG 6-Thioguanine - TT Thiotepa - UV Ultraviolet light - V_f Variant frequency     

Estimation by limiting dilution analysis of human IL 2-secreting T cells: detection of IL 2 produced by single lymphokine-secreting T cells

We present here a culture method for the estimation, in human blood, of the number of lymphocytes that can respond to mitogen by producing interleukin 2 (IL 2). T cells are cultured at limiting dilutions with PHA or Con A in the presence of Epstein Barr virus-transformed human lymphoblastoid cells (EB-LCL), and supernatants are tested 3 days later for IL 2 content by a cell proliferation assay. The distribution of negative wells follows the expected Poisson "single-hit" relationship, suggesting that the assay is sensitive to single cells of a single limiting cell type. On average, 16.3% of peripheral blood mononuclear cells can produce IL 2 in such clonal cultures (mean of 12 determinations; SD = 5.6%). Surprisingly, irradiation (up to 2000 rad) of the titrated responder cell population diminishes the estimated frequencies by less than 50%. The ability to detect IL 2 levels in cultures containing only a single, nonproliferating T lymphocyte allows us to estimate the amount of IL 2 generated by an individual effector cell during a 3-day culture interval after mitogen stimulation. The average responding, irradiated T cell generates 0.92 pg of IL 2 (median) within 3 days. The method presented provides a straightforward way to provide independent estimates of responding cell number and of lymphokine production per cell in a variety of clinical situations.     

Presence of a reversible inhibitor for human lymphoid cell DNA synthesisin the extracts of human peripheral lymphocytes.

An endogenous inhibitor of human lymphocyte DNA synthesis contained in extracts of purified human peripheral lymphocytes is described. It was found that the peripheral lymphocyte extract inhibits the DNA synthesis of phytohemagglutinin (PHA) stimulated human peripheral lymphocytes, lymphocytes in mixed lymphocyte culture, and human lymphoid cells in a long-term culture (PGLC-33H). This extract did not inhibit the DNA synthesis of nonlymphoid cells including HeLa and human embryonic lung. The effects of the inhibitor were reversible and noncytotoxic. Initial characterization showed the inhibitor to be thermolabile, DNase resistant, trypsin sensitive, and stable in a pH range 5.4–8.4. It appears that the inhibitor contained in the purified human peripheral lymphocyte extract is similar to a previously described inhibitor extracted from a human lymphoid cell line (PGLC-33H). Quantitation of the inhibitor in various lymphoid cell populations showed the amount of inhibitor per cell to be higher in resting peripheral lymphocytes than in PHA stimulated peripheral lymphocytes or human lymphoid cells in long-term culture (PGLC-33H). This data suggest that the inhibitor described may play a regulatory role in lymphocyte metabolism.     

Enumeration of 6-thioguanine-resistant peripheral blood lymphocytes in man as a potential test for somatic cell mutations arising in vivo.

An autoradiographic method to enumerate variant 6-thiogunanine-resistant (TGr) peripheral blood lymphocytes (PBLs) that occur in vivo in man is described. Variant cells are detected in PBL cultures stimulated to tritiated thymidine (3HTdr) incorporation in vitro with phytohemagglutinin (PHA) in the presence of TG. Cells with the naturally-occurring Lesch--Nyhan (LN) mutation served as prototype-variant cells. PBLs from a LN hemizygous male were found to be resistant to TG inhibition of PHA-stimulated 3HTdr in corporation in vitro while a LN heterozygous female was found to be a mosaic with 2/1000 PBLs resistant to 2 X 10(-4) M TG. Experiments with artificial mixtures of LN and normal PBLs showed that the LN cells were virtually all detectable even when present in low frequency (10(-5)). TGr PBLs were found in healthy non-LN individuals at median frequencies of 1.0 X 10(-4) and 1.1 X 10(-4) when determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG respectively. Their frequencies were not age-related. TGr PBL-variant frequencies (Vf's) were determined in 47 cancer patients who were being treated with cytotoxic agents that are known to be mutagens. The median TGr PBL Vf determined at 2 X 10(-3) M TG in cancer patients was 2.2 X 10(-4) while, when determined at 2 X 10(-4) M TG, it was 8.5 X 10(-4). The distribution of Vf's for the treated cancer-patient group differed from that for the normal control group in that more than half of the treated cancer patients had TGr PBL Vf's greater than the highest seen for controls. Unlike those of the normal controls, the TGr PBL Vf's of treated cancer patients differed if determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG, a behavior that suggested partial resistance and mimicked that seen with LN TGr PBLs. PBLs resistant to 2,6-diaminopurine (DAPr) were not found in two individuals, although the TGr PBL Vf was elevated in one. TGr PBL Vf's were greatly elevated under conditions of in vivo selection in patients receiving purine-analogue immunosuppressive therapy. The TGr PBL enumerative assay system is presented as one of potential value to detect somatic cell mutations occurring in vivo in man.