The Binding of Cations to the Surfaces of Cells from Early Chick Blastoderms

Cells of the early chick blastoderms are either preparing for or undergoing regulated morphogenetic movements which culminate in the formation of a three-layered embryo. Information on the changes in the physical-chemical properties of cell surfaces may help in the understanding of this process. The binding of magnesium, manganese, strontium, barium and lanthanum to surfaces of early embryonic cells was estimated by the changes induced by these cations in the cells' electrophoretic mobilities (EPM). Cells show a positive EPM at concentrations of MgCl₂ and MnCl₂ at 3 × 10⁻² M while SrCl₂, and BaCl₂ were not able to reverse the cells' charge at concentrations up to 6 × 10⁻² M. CaCl₃ reversed the cells' EPM at concentrations as low as 5 × 10⁻³ M.
Our results suggest that the surfaces of early embryonic cells have a high affinity for Mg and Mn. This is indicated by a reversal of polarity which cannot be detected in cells of differentiating or adult tissues at the cation concentrations used in these experiments.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin reduces high-affinity binding of epidermal growth factor to cell surface receptors in C3H 10T1/2 cells

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF)-binding characteristics was studied in a cultured embryonic fibroblast cell line, C3H 10T1/2. At very low concentrations, TCDD was found to cause a persistent decline in EFG binding, the median effective concentration (EC-50) being 10(-12) M. This particular effect was most conspicuous when TCDD was added at the time of medium change with fresh Dulbecco's modified Eagle's medium. Cells at an early stage of confluency were more responsive to TCDD than those at a later stage. Although most reported TCDD-evoked biological changes are recognized to occur slowly during the course of a few days to weeks, the response of C3H 10T1/2 cells to TCDD was swift, showing a sign of decline of EGF binding as early as three hours after TCDD addition. C3H 10T1/2 cells appear to be an excellent in vitro model to study TCDD's biochemical action mechanisms.     

Effect of surface-active agents on the electrical properties of bacterial cells

The work was aimed at studying the effect of cationic, anionic and non-ionogenic surfactants on the frequency dependence of the electroorientation effect (EOE) and on the electrophoretic mobility (EPM) of rod-like bacteria. The character of concentration dependences was found to differ for EOE and EPM at a low frequency of the electric field (20 to 10(4) Hz). Analysis of EOE changes at a high frequency (4 X 10(5) to 3 X 10(7) Hz) showed that anionic and non-ionogenic surfactants at a concentration up to 10(-3) M did not damage Escherichia coli, a Gram-negative bacterium, in contrast to Bacillus cereus, a Gram-positive bacterium. Cationic surfactants affected the cells of the both species. The optical properties of bacterial cells were found to change under the action of cationic surfactants.     

Clastogenic effects of methylnitrosourea and ethylnitrosourea on chromosomes from human fibroblast cell lines.

Human fibroblast cell lines were pulse-treated for 1 h with either methylnitrosourea (MNU) or ethylnitrosourea (ENU) at various time intervals before harvesting for chromosome analysis. Cells treated with 1 X 10(-3) M, 5 X 10(-4) M, and 1 X 10(-4) M final concentrations of MNU and ENU during the G2 or M phases of the cell cycle showed a significant increase in chromatid-type abnormalities over controls. Cells exposed to MNU or ENU 23 h before harvest showed some chromosome-type abnormalities, reflecting probable damage induced during the G1 phase of the cell cycle or derived from chromatid damage induced during the previous cell cycle. The mitotic indices and incidences of abnormalities suggested a dose response effect when cells were treated with the two higher concentrations and the three concentrations, respectively, of MNU or ENU. Chromatid abnormalities were observed in MUN and ENU-treated cells from each of four cell lines. From this investigation, it was concluded that MNU and ENU treatment of human diploid cell lines in vitro induced both chromatid and chromosome aberrations. MNU and ENU, both of which had previously been shown to be mutagenic in experimental animals, are, therefore, also considered to be mutagenic at the chromosome level in human fibroblasts grown and treated in cell culture.     

Stimulation of 86Rb+ and 32Pi movements in 3T3 cells by prostaglandins and phorbol esters

The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent.     

Modulating effects of phenothiazines on mast cells deformities induced by colchicine. Importance of calcium

The shape changes of peritoneal rat mast cells induced by colchicine are completely inhibited by trifluoperazine (10(-4) M), known to inhibit calmodulin, and by EDTA (2 X 10(-3) M). Promethazine and chlorpromazine increase these modifications up to 10(-4) M and inhibit them at higher concentrations.     

2,3,7,8-tetrachlorodibenzo-p- dioxin reduces high-affinity binding of epidermal growth factor to cell surface receptors in C3H 10T1/2 cells

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF)-binding characteristics was studied in a cultured embryonic fibroblast cell line, C3H 10T1/2. At very low concentrations, TCDD was found to cause a persistent decline in EFG binding, the median effective concentration (EC-50) being 10^?12M. This particular effect was most conspicuous when TCDD was added at the time of medium change with fresh Dulbecco's modified Eagle's medium. Cells at an early stage of confluency were more responsive to TCDD than those at a later stage. Although most reported TCDD-evoked biological changes are recognized to occur slowly during the course of a few days to weeks, the response of C3H 10T1/2 cells to TCDD was swift, showing a sign of decline of EGF binding as early as three hours after TCDD addition. C3H 10T1/2 cells appear to be an excellent in vitro model to study TCDD's biochemical action mechanisms.     

Immunomodulatory effects of corticosteroids on natural killer and antibody-dependent cellular cytotoxic activities of human lymphocytes

The in vitro effect of prednisolone (PRD) on NK and ADCC activities of human lymphocytes was investigated. PRD at concentrations ranging from 7.5 X 10(-3) to 1 X 10(-5) M significantly inhibited NK activity, while concentrations of 7.5 X 10(-3) to 1 X 10(-4) M inhibited ADCC activities of PBL when added directly to the mixture of effector and target cells. Lymphocytes pre-cultured for 24 hr with PRD at concentrations ranging from 1 X 10(-4) M to 1 X 10(-6) M showed significant suppression of their NK activity. Inhibition was proportional to the concentration of the drug, and was observed at as early as 1 hr of incubation at various effector to target cell ratios with several targets. PRD also inhibited NK and ADCC activities of purified T cells, non-T cells, and NK-enriched effector cells. In target-binding assays, PRD decreased the target-binding capacity of effector lymphocytes in a dose-dependent manner. PRD-induced inhibition could be reversed by incubating lymphocytes for 1 hr with interferon or IL 2. Pretreatment of targets with PRD for 4 hr did not affect cytotoxic activity. Inhibition of cytotoxicity was not due to direct toxicity to effector cells because lymphocytes treated with PRD showed normal spontaneous 51Cr release, and their viability after 24 hr of pre-culture with PRD was comparable to that of untreated control cells. These results demonstrate that PRD has significant immunomodulatory effects on human NK and ADCC activities that may be of clinical relevance.     

Effect of mercuric chloride and methylmercury on photosynthetic activity of the diatom Thalassiosira weissflogii by a fluorescent method

It was shown by the pulse-amplitude modulation fluorescent method that, at a weak illumination (6 microE m-2.s-1), methylmercury at a concentration of 10(-6)-10(-7) M decreases the photochemical activity of the reaction centers of photosystem II in cells of microalgae Thalassiosira weissflogii after a prolonged lag phase. Cells resistant to methylmercury at these low concentrations were detected by the microfluorimetric method. Chloride mercury decreased the activity of photosystem II of the algae only when at higher concentrations. Both toxicants at a concentration of 10(-6) M decreased the rate of recovery of photoinduced damage of centers of photosystem II and led to an increase in the energization component of nonphotochemical fluorescence quenching. These results indicate that the complex of fluorescent methods can be used to monitor early changes in the photosynthetic apparatus of algae in response to the toxic action of heavy metals.     

Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The ability of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp cAMPS) to inhibit glucagon-induced glycogenolysis was studied in hepatocytes isolated from fed rats. Preincubation of the cells for 20 min with progressively higher concentrations of Rp cAMPS followed by a 1 X 10(-9) M glucagon challenge resulted in a 50% inhibition of glucose production over a 30-min period at 2-3 X 10(-6) M Rp cAMPS. A maximal inhibition of 50-74% was achieved, the actual value depending upon the length of preincubation with Rp cAMPS. The inhibitory effect did not increase when the concentration of Rp cAMPS was increased from 3 X 10(-6) to 3 X 10(-4) M. Addition of 1 X 10(-5) M Rp cAMPS to the cells followed by 10(-11) to 10(-6) M glucagon shifted the glucagon concentration required for half-maximal glucose production measured at 10 min to 6-fold higher glucagon concentrations and the concentration of glucagon required for apparent maximal glucose production measured at 10 min to greater than 10-fold higher glucagon concentrations. The cAMP-dependent protein kinase activation curve was similarly shifted to higher concentrations of glucagon. These data show that Rp cAMPS acts as a cAMP antagonist capable of opposing the glucagon-induced activation of cAMP-dependent protein kinase and the concomitant activation of the glycogenolytic cascade.     

Dose-rate effects of 8-methoxypsoralen plus 365-NM irradiation on cell killing in Saccharomyces cerevisiae.

Tow types of dose-rate effect that alter the survival response of haploid yeast cells to 8-methoxypsoralen (8-MOP) plus treatment with irradiation at 365 nm were studied. (1) When the concentration of 8-MOP was varied between 9.2 X 10(-5) and 2.3 X 10(-8) M and the dose rate of 365-nm irradiation kept constant, the efficiency of the irradiation for killing increased relatively to that of 8-MOP whe the concentration of 8-MOP decreased. This indicated that there was no strict reciprocity between radiation dose and concentration of drug. (2) When the dose rate of radiation was varied between 0.66 X 10(3) and 108 X 10(3) J m-2 h-1 and the concentration of 8-MOP was kept constant, the survival of wild-type cells increased strikingly at low dose rates of radiation as compared with high dose rates. Cells responded more to changes at low dose rates than to equal changes a high dose rates. The high resistance of wild-type cells to 8-MOP plus radiation delivered at low dose rates absent from rad 1-3 cells defective in excision-repair. This suggests that the dose-rate effect seen in wild-type cells depended at least in part on an active excision-repair function. At low dose rates of radiation, the shoulder of the survival curve for rad1-3 cells, i.e. the ability to accumulate sub-lethal damage, was increased by a factor of about 2 when compared with that seen at a high dose rate. Thus it is likely that at low dose rates a repair function other than excision-resynthesis may operate in rad1-3 cells.     

Respiratory resistance of methylotrophic bacteria to formate and cyanide

Whole cells and cell-free preparations of the methylotrophic bacteria, Pseudomonas sp. AM 1 and Achromobacter parvulus, can oxidize formate at tis concentration in the reaction medium up to 1 M. The respiration of whole cells is registered at a concentration of formate greater than 10(-2) M, while that of cell-free extracts at a formate concentration greater than 5 X 10(-5) M. This seems to be due to the presence of a permeability barrier in cells for formate. The oxidation of reduced TMPD and exogenous cytochrome c by the membrane preparations of the two bacteria is inhibited by formate and cyanide; Ki50% = 2.5 X 10(-2) and 10(-6) M, respectively. The oxidation of NADH by the membrane preparations of the bacteria is not inhibited by 1 M formate and 5 X 10(-4) M cyanide but is inhibited by formaldehyde with Ki50% = 3 X 10(-2) M. Formaldehyde has no effect on the oxidation of reduced TMPD and cytochrome c at concentrations greater than 2 X 10(-1) M. These data indicate that respiration of the studied methylotrophic bacteria in the presence of high formate concentrations should be attributed in the presence of a branched electron transport chain in them; one branch of the chain is resistant to formate and cyanide, but is sensitive to formaldehyde.     

Prostaglandin inhibition of testosterone production induced by luteinizing hormone, dibutyryl cyclic AMP or 3-isobutyl-1-methyl xanthine in dispersed rat testicular interstitial cells.

Testicular interstitial cells were utilized to study the effects of prostaglandins (PG) on in vitro testosterone production and to examine the role of cyclic adenosine-3',5'-monophosphate (cAMP) in this process. Testosterone production was assessed after 3 hour incubations while cAMP accumulation was examined after a 0.5 hour incubation period. Testosterone and cAMP were measured by radioimmunoassay. None of the PGs tested (PGA, PGA2, PGB1, PGE1, PGE2, PGF1alpha PGF2alpha) altered basal testosterone production when present in incubates at concentrations of 1.3 X 10(-8) M to 1.3 X 10(-4). However, at concentrations of 1.3 X 10(-4) M all of these PGs were capable of decreasing Luteinizing Hormone (LH; 100ng)-induced testosterone production. The inhibition of LH-induced testosterone production by the B, E and F series PGs was less pronounced than that for the A series. PGA1 and PGA2 exhibited 80% and 95% inhibition, respectively, at 1.3 X 10(4) M. The inhibitory action of 4 X 10(5) M PGA1 or PGA2 was evident within 30 minutes. Preincubation of interstitial cells with indomethacin (10(-5) or 10(-6) M) for 30 minutes did not alter subsequent basal or LH (100ng)-induced testosterone production. Accumulation of cAMP was stimulated by LH (10 microgram) or by PGs (1.3 X 10(-4) M PGA1, PGA2, PGB1, PGE1 or PGF2alpha). The PG-induced cAMP accumulation thus occurred at concentrations where LH-stimulated testosterone production was inhibited. Furthermore, PGA1 and PGA2 (1.3 X 10(-4) M) inhibited testosterone production induced by either 3-isobutyl-1-methyl xanthine (MIX; 10(-4) M or 10(-3) M) or dibutyryl cAMP (dbcAMP; 10(-4) M or 10(-3) M). These results indicate that PGs can block testosterone production by a direct effect on testicular interstitial cells and suggest that PGs exert their inhibitory action distal to stimulation of cAMP formation. PGs do not appear to play a role in the mechanism of LH action.     

Calcium-dependent process in reduction of cell surface charge after x-irradiation.

The electrophoretic mobility (EPM) of rat erythrocytes and cultured melanoma cells decreased with time after X-irradiation in the presence of calcium at concentrations higher than 10 (-5) M. At 37 degrees C, the presence of calcium for the first 20 min of exposure was suffcient to induce the EPM reduction, and Ca 2+ administration subsequent to Ca 2+ -free incubation for 30 min following irradiation had no effect on EPM. At lower temperatures, from 10 down to 20 degrees C however, the effect of calcium on the reduction of EPM decreased drastically. If the cells were kept Ca 2+ -inonophore A23187 also induced to decrease in EPM only in the presence of Ca 2+. These results revealed the transitory existence of membrane condition reactive to extracellular Ca 2+ immediately after X-irradiation, which can be postponed at low temperatures. The reduction of EPM by Ca 2+ -ionophore might suggest that the influx of Ca 2+ is a step in the reduction of EPM after X-irradiation.     

Selectivity of hemoregulatory peptide (HP5b) action in culture

A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.     

Combined expression of different hormone genes in single cells of normal rat and mouse pituitary

Cells displaying combined expression of different pituitary hormone genes (further referred to as 'multi-hormone mRNA cells') were identified in normal rat and mouse pituitary by single cell RT-PCR. These cells do not seem to produce or store all the respective hormones the mRNAs encode for. The cells are already developed at day 16 of embryonic life (E16) in the mouse. Different peptides, such as gamma3-melanocyte-stimulating hormone (gamma3-MSH) and gonadotropin-releasing hormone (GnRH), affect different subsets of these cells. In culture, estrogen and GnRH increase the number of 'multi-hormone mRNA cells' that contain prolactin (PRL) mRNA or mRNA of the alpha-subunit of the glycoprotein hormones (alpha-GSU) but not the number of 'multi-hormone mRNA cells' not containing PRL or alpha-GSU mRNA. 'Multi-hormone mRNA cells' may function as 'reserve cells' in which a particular hormone mRNA may be translated under a particular physiological condition demanding a rapid increase of that hormone.     

Excitation-contraction coupling in cardiac Purkinje fibers. Effects of cardiotonic steroids on the intracellular [Ca2+] transient, membrane potential, and contraction

The [Ca2+]-activated photoprotein aequorin was used to measure [Ca2+] in canine cardiac Purkinje fibers during the positive inotropic and toxic effects of ouabain, strophanthidin, and acetylstrophanthidin. The positive inotropic effect of these substances was associated with increases in the two components of the aequorin signal, L1 and L2. On the average, strophanthidin at 10(-7) M produced steady, reversible increases in L1, L2, and peak twitch tension of 20, 91, and 240%, respectively. This corresponds to increases in the upper-limit spatial average [Ca2+] from 1.9 X 10(-6) M to 2.1 X 10(-6) M at L1 and from 1.4 X 10(-6) M to 1.8 X 10(-6) M at L2. Elevation of diastolic luminescence above the control level was not detected. At higher concentrations (5 X 10(-7) M), strophanthidin produced aftercontractions, diastolic depolarization, and transient depolarizations, all of which were associated with temporally similar changes in [Ca2+]. During these events, diastolic [Ca2+] rose from the normal level of approximately 3 X 10(-7) M up to 1-2 X 10(-6) M. The negative inotropic effect of 5 X 10(-7) M strophanthidin was not associated with a corresponding decrease in the [Ca2+] transient but was associated with a change in the relationship between [Ca2+] and tension. Assuming the Na+-lag mechanism of cardiotonic steroid action, we conclude the following: at low concentrations of drug, increased Ca2+ uptake by the sarcoplasmic reticulum prevents a detectable rise in cytoplasmic [Ca2+] during diastole, but this increased Ca2+ uptake results in increased release of Ca2+ during the action potential. At higher drug concentrations, observable [Ca2+] changes during diastole activate tension and membrane conductance changes.     

Possible mechanisms of positive inotropic action of synthetic human calcitonin gene-related peptide in isolated rat atrium

The effect of synthetic human calcitonin gene-related peptide (hCGRP) on the isolated and electrically driven left atria of rats were investigated. The peptide at concentrations of 3 X 10(-9)-3 X 10(-7) M produced positive inotropic effects on the left atria in a dose-dependent manner. Verapamil (10(-5) M) and adenosine (10(-4) M) reduced the positive inotropic effect of hCGRP at concentrations of 3 X 10(-9) and 3 X 10(-8) M, but not at that of 3 X 10(-7) M. Ouabain (5 X 10(-5) M) inhibited the effect of hCGRP in concentrations of 3 X 10(-7) and 3 X 10(-8) M, but not in that of 3 X 10(-9) M. Simultaneous pretreatment with verapamil (10(-5) M) and ouabain (5 X 10(-5) M) suppressed the positive inotropy by hCGRP at all concentrations tested. On the other hand, tetrodotoxin (10(-6) M) potentiated only the positive inotropic effect of 3 X 10(-7) M hCGRP. Metoprolol (10(-7) M) and theophilline (10(-3) M) did not affect the inotropic effect of hCGRP. These results suggest that the positive inotropic effect of hCGRP is not mediated by beta-adrenoceptors but by two distinct mechanisms of action, which was inhibited by verapamil but not by ouabain (facilitation of Ca++ influx in lower concentrations of hCGRP) and which was blocked by ouabain but not by verapamil and potentiated by tetrodotoxin (inhibition of Na+/Ca++ exchange mechanism at higher concentrations of hCGRP).     

NMR investigation of the influence of procaine and its metabolites on the water exchange through human erythrocyte membranes

The effect of procaine hydrochloride and its metabolites on the diffusional water exchange through erythrocyte membranes was investigated at 37 degrees C and at concentrations ranging between 5 X 10(-5) M and 5 X 10(-1) M by using the NMR manganese doping method. Procaine hydrochloride and 2-diethylaminoethanol have a moderate stimulating effect on the water exchange, of up to 20% at concentrations ranging between 10(-3) and 10(-2) M, while an increasing inhibitory effect was found at higher concentrations. The p-aminobenzoic acid has no effect on the water exchange up to 10(-2) M and, at higher concentrations, and apparent decreasing inhibition was noticed which is thought to be an artefact due to the uptake of Mn2+ by the cells. The temperature dependence studies suggest that procaine HCl enhances the uptake of Mn2+ by the cells. An opposite effect was found for rigid erythrocytes. The p-aminobenzoic acid and 2-diethylaminoethanol appeared to be more effective than procaine hydrochloride in increasing the uptake of Mn2+.     

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.