CI Solvent Yellow 14 shows activity in the bone marrow micronudeus assay in both the rat and mouse

CI Solvent Yellow 14 has been reported to be carcinogenic inthe rat, inducing neoplastic liver nodules, but noncarcinogenicin the mouse. The present experiments have extended previouslyreported investigations on the activity of CI Solvent Yellow14 in in vivo genotoxicity assays. CI Solvent Yellow 14 hasbeen examined for genotoxicity in vivo in the bone marrow micronudeusassay in both the rat and the mouse, and in the rat liver unscheduledDNA synthesis (DNA repair) assay, to limit doses of 5000 and2000 mg/kg respectively, by oral gavage. CI Solvent Yellow 14showed evidence of clastogenic activity in both the rat andmouse bone marrow (clear effect in the rat; weak effect in themouse), but no evidence of DNA repair in the rat liver. In viewof the latter finding, the contribution, if any, of the genotoxicityexpressed by the micronucleus assay to the formation of livernodules on chronic administration of the compound, is unclear. ³To whom correspondence should be addressed     

AT base pairs are the main target for mutations at the hprt locus of rat skin fibroblasts exposed in vitro to the monofunctional alkylating agent N-ethyl-N-nitrosourea

Spectra of N-ethyl-N-nitrosourea (ENU)-induced mutations differwidely among various in vitro and in vivo mutational systems.To investigate possible reasons for these differences, a mutationalsystem is needed in which the same target gene is used for comparisonin the same type of cells in vitro and in vivo. In the presentstudy, this was achieved by analysing at the molecular level35 hprt mutant rat fibroblast clones obtained from cell populationsexposed in vitro to ENU and comparing the mutational spectrumwith the previously determined spectrum of ENU-induced hprtmutants in the same target cells exposed in vivo. Twenty-eightmutants contained a single base pair alteration in the hprtcoding sequence. Most of these changes were found at AT basepairs (19/28), the AT to TA transversion being the most frequentkind of mutation (12/19), which is probably caused by O²-ethylthymine.Transversions at AT base pairs showed all mutated T's to belocated in the nontranscribed strand of the hprt gene, suggestinga strand specific fixation of mutations induced by O²-ethylthymine,which appears to be a general feature of ENU- and ENNG-inducedhprt mutations in mammalian cells. GC to AT transitions, probablycaused by O⁶-ethylguanine, were detected at a lower frequency(7/28). This in vitro mutational spectrum was very similar tothat of the same target cells exposed in vivo to ENU. A comparisonof the mutational spectra in AGT-proficient and AGT-deficientrodent cells exposed to ethylating agents showed that in contrastto the situation in AGT-proficient rat fibroblasts, GC to ATbase pair changes (and not AT to TA) are the predominant mutationsin AGT-deficient hamster cells. ⁴To whom correspondence should be addressed     

Assessment of the potential in vivo genotoxicity of three trihalomethanes: chlorodibromomethane, bromodichloromethane and bromoform

Chlorination of drinking water results in the formation of chlorodibromomethane,bromodichloromethane and bromoform. These trihalomethanes haveall shown evidence of genotoxicity in bacterial and mammaliancell systems in vitro and some evidence of carcinogenicity inrodents. Chlorodibromomethane and bromodichloromethane havepreviously been tested in the mouse micronucleus test and didnot induce chromosome damage, but results from two previousmicronucleus tests on bromoform are somewhat contradictory.In the present study, bromoform was tested in the mouse bonemarrow micronucleus test in order to reassess the response inthis system; all three compounds were evaluated using the ratliver unscheduled DNA synthesis test. Trihalomethanes are wellabsorbed by the oral route which was selected for this studyas being that most relevant to humans. Bromoform did not inducemicronuclei in mouse bone marrow, and chlorodibromomethane,bromodichloromethane and bromoform did not cause unscheduledDNA synthesis in rat liver. These trihalomethanes have not shownany evidence of genotoxicity in vivo and are most unlikely tohave any significant genotoxic activity in mammals. Their modeof action as rodent carcinogens remains unexplained. ¹To whom correspondence should be addressed     

Fas (CD95)-independent regulation of immune responses by antigen-specific CD4 CD8+ T cells

Antigen-activated T cells of the CD4⁺CD8^– and the CD4^–CD8⁺phenotype are susceptible to antigen receptor-stimulated celldeath. This form of apoptotic cell death has been shown to bedependent on the expression of the Fas (CD95) antigen and canoccur via an autocrine mechanism involving the concomitant up-regulationof Fas and its ligand on activated T cells. Mutations in genesencoding Fas (lpr) and the Fas ligand (gld) contribute to thedevelopment of an autoimmune syndrome similar to systemic lupuserythematosus in mice. These observations led to the suggestionthat the Fas signaling pathway is an important regulator ofimmune responses in vivo. Here we evaluated the importance ofthe Fas pathway in regulating immune responses by male antigen-specificCD4^–CD8⁺ T cells. We found that the in vivo eliminationof male antigen-activated cells was independent of Fas expressionby these cells. However, the elimination of these activatedcells was inhibited by the transgenic expression of Bcl-2, aprotein that inhibits multiple forms of apoptotic cell death.The transgenic Bcl-2 protein also inhibited the death of maleantigen-activated cells following IL-2 deprivation. Cell deathresulting from IL-2 deprivation occurred efficiently in maleantigen-activated Fas^- cells. We propose that the rapid deletionof male antigen-activated Fas^– cells in vivo is due tolimiting amounts of IL-2 that are available in the microenvironmentof the activated cells at the peak of the response.     

Mutagenicity testing of rutacridone epoxide and rutacridone, alkaloids in Ruta graveolens L., using the Salmonella/microsome assay

The mutagenicity of rutacridone and rutacridone epoxide wasinvestigated using Salmonella typhimurium without as well aswith different metabolic activation systems. Rutacridone epoxidewas found to be a direct acting mutagen in S. typhimurium strainsTA98, TA100 and TA1538; addition of rat liver preparations resultedin a marked decrease of mutagenicity. In contrast, rutacridonerequired metabolic conversion to exhibit mutagenic activity.Neither of the compounds had any effect on tester strain TA1978.S9 mixes as well as microsomal and cytosolic preparations fromuntreated, phenobarbital-treated and 3-methylcholanthrene-treatedrats were used to study the activation and deactivation capacitiesof the enzyme mixtures. In addition, the influence of enzymeinhibitors on the activation and deactivation of the furoacridoneswere tested. Evidence is presented that rutacridone is metabolizedby rat liver enzymes to the corresponding epoxide as the ultimatemutagen. ^*Dedicated to Professor Dr C.-G.Arnold on the occasion of his60th birthday.      

A pivotal role of IL-12 in Th1-dependent mouse liver injury

Intravenous injection of Proplonibacterium acnes and llpopolysaccharide(LPS) with a 7 day interval caused CD4⁺ T cell-dependent severeliver injury in the C57BL/6 (H-2^b) mouse strain. In contrast,BALB/c (H-2^d mice were resistant to P. acnes and LPS-inducedliver injury. The different susceptibilities of the two mousestrains to liver injury appeared to be closely correlated withtheir different abilities to produce IFN- after P. acnea priming.Namely, the sensitive C57BL/6 mouse strain produced a significantlevel of IFN- 7–10 days after P. acnes injection, whereasno significant amount of serum IFN- was detected in the resistantBALB/c mouse strain. The important role of IFN- in liver injurywas demonstrated from the finding that In vivo administrationof anti-IFN- mAb abrogated P. acnes and LPS-induced liver injuryin C57BL/6 mice. Moreover, it was demonstrated that In vivoadministration of recombinant IL-12, a key cytokine for theinduction of IFN-, into mice induced P. acnes and LPS-inducedliver injury in the resistant BALB/c mouse strain. Conversely,In vivo administration of anti-IL-12 mAb blocked the developmentof liver injury in the sensitive C57BL/6 mouse strain. Moreover,it was demonstrated that the failure of the induction of liverinjury in BALB/c mice appeared to be derived from the lack ofexpression of IL-12 at the local site of liver in P. acnes-prlmedmice. These results strongly indicated that endogenous IL-12,which stimulates T^h 1-dominant cellular immunity and IFN- production,may be an essential cytokine on the course of T cell-dependentliver injury.     

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo

Clonal deletion and/or inactivation establishes tolerance toself antigens. Endogenous and exogenous (bacterial) superantigens,like the staphylococcal enterotoxlns, induce ligand-specificclonal anergy in vivo and thus are believed to mirror aspectsof post-thymic tolerance mechanisms in mature peripheral T cells.Here we analyzed the level of anergy of ligand-responsive V_ß8⁺T cells from staphylococcal enterotoxin B (SEB)-primed micein vivo and in vitro. Upon in vitro restimulation with SEB,CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells failed toproduce IL-2. However, functional IL-2 receptors were triggered,since supplementation with IL-2 induced clonal growth in virtuallyall CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells as determinedby limiting dilution analyses. Thus in vitro unresponslvenessof lymphocytes from SEB-primed mice reflects the inability ofSEB-reactlve V_ß8⁺ T cells to produce IL-2. Surprisingly,anergy as defined in vitro was at variance with that in vivo.Following further challenge with SEB, systemic and acute lymphokineproduction (Including IL-2 and tumor necrosis factor) occurredwith almost identical peak values and kinetics to primary invivo responses, and D-galactosamlne-sensltlzed mice succumbedto lethal shock. Polymerase chain reaction analyses revealedthat CD4⁺V_ß8⁺ expressed IL-2-specific mRNA in vivoupon restimulatlon with SEB. While lymphokine production andexpression of the IL-2 receptor was similar to the responseto in vivo primary stimulation, only CD8⁺V_ß8⁺ T cellsexpanded clonally upon reintroductlon of SEB in vivo. Henceprimed V_ß8⁺ T cells challenged with SEB display invitro anergy yet in vivo responsiveness, at least in part. Weconclude that the state of anergy is reversible, dependent uponthe quality of activation signals provided in in vivo ratherthan in in vitro culture conditions.     

DNA sequence analysis of spontaneous mutations at a LacZ transgene integrated on the mouse X chromosome

Transgenic mice with integrated shuttle vectors containing theLacZ mutational target gene were used to study spontaneous mutationalevents in vivo. The transgenic mouse strain used carries theLacZ transgene on the X chromosome and was previously foundto be characterized by 25–fold higher spontaneous mutationfrequency in liver and brain compared with at least three othertransgenic mouse strains. To determine the nature of in vivospontaneous mutational events, 35 mutant LacZ genes isolatedfrom liver and brain of mice from strain 35.5 were analyzedat the DNA sequence level. The results obtained indicate thatsingle base-pair changes were predominant in both liver andbrain. However, in liver the majority of mutations were transitionswhereas in brain transversions were predominantly observed.Six mutants appeared to contain multiple dispersed mutations,separated by as much as 44 bp. Mutations were generally locatedwithin a 500 bp region encoding the active site of the ß-galactosidaseprotein. Our results indicate that spontaneous mutations atthe LacZ transgene are tissue specific and dependent on thechromosomal position of the LacZ transgene. ³To whom correspondence should be addressed at: Harvard Medical School, Beth Israel Hospital, 330 Brooldine Avenue, Boston, MA 02215, USA     

The w/w+ SMART is a useful tool for the evaluation of pesticides

Genotype-dependent variability in the response of several Drosophilastrains to hexamethylphosphoramide (HMPA) has been studied usingthe white/white⁺ (w/w⁺) somatic mutation and recombination test(SMART). Among the tester strains, there were three wild-typelaboratory strains (Leiden-S, Oregon-K and 91-C) and three insecticide-resistantstrains (Haag-79, Hikone-R and 91-R). The response to HMPA oflarvae from a cross between two wild-type strains (Leiden-Sand Berlin-K) was also measured. The strains have been evaluatedin terms of spontaneous frequencies of mosaic eyes, lowest effectivedose and dose—response relationship. Strong variabilitywas found among the strains, the best performance to HMPA beingobtained with the strain Oregon-K. In addition, a series ofpesticides structurally related to HMPA, such as dimefox, hexamethylmelamine,hexazinone, alachlor, CAM, pirimicarb, dimetilan, thiram andmethabenzthiazuron have been tested with the Oregon-K strain.Some of these pesticides had already been shown to be genotoxicin other systems, whereas others have either not been testedor gave negative results in in vitro systems. Although genotoxicitywas expressed only within a narrow dose range, all pesticideswere genotoxic in the w/w⁺; system with the Oregon-K strain.Thus, these compounds may be a genotoxic hazard to man. Theseresults suggest the suitability of the strain Oregon-K for genotoxicitytesting with the w/w⁺ eye mosaic system, although more informationabout the performance of this strain with other compounds mustbe obtained. It is concluded that the w/w⁺ SMART is an excellentin vivo system able to reveal genotoxicity of promutagens thatare difficult to detect with in vitro genotoxicity test systems.     

Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus

Listeria monocytogenes spends most of its intracellular lifecycle in the cytosol of the infected eucaryotic cells. Withinthis cellular compartment originates the endogenous pathwayof antigen processing and presentation. We thus assumed thatrecombinant L. monocytogenes expressing an heterologous protein,the nucleoprotein of the lymphocytic choriomeningltis virus(LCMV), should be able to induce antigen-specific CD8⁺ T cellsin vivo. The LCMV nucleoprotein gene was inserted in phase withthe sequence coding for the putative signal sequence of thehemolysin of L. monocytogenes in order to target its secretioninto the cytosol of the infected cell. The ability of this recombinantbacterium to induce LCMV-reactive CD8⁺ T cells was then monitoredin BALB/c mice. The immune status of the immunized BALB/c micewas studied on the seventh day after a single i.v.injectionof a sublethal dose of the recombinant bacteria: (i) cytotoxicCD8⁺ T cells were detected in liver; (ii) using in vitro re-stimulationwith PMA and ionomycin, secondary cytotoxic CD8⁺ T cells weredetected in spleen; (iii) an early inflammatory reaction dependenton the presence of CD8⁺ T cells occurred in the footpad afterintraplantar inoculation of live LCMV; and (iv) mice were protectedagainst an otherwise lethal intracerebral LCMV challenge; theprotection was accompanied by elimination of the virus. Whenthe immune status of the immunized hosts was monitored for alonger period post-immunization, the balance between immuneprotectiosn and immunopathology described for the anti-LCMVimmune responses was observed; two phases of protection weredetected, flanking a transitory phase of exacerbation of thelymphocytic choriomeningitis disease (weeks 2–5). Takentogether, these results indicate the feasibility of using attenuatedL. monocytogenes as a model of a live vector to induce in vivoCD8-ependent immune responses against intracellular pathogens.     

Intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 are involved in protection mediated by CD3+TCR{alpha}{beta} T cells at the early stage after infection with Listeria monocytogenes in rats

To Investigate the significance of Intercellular adhesion molecule-1(ICAM-1) and leukocyte function-associated antlgen-1 (LFA-1)In host defense against infection with Intracellular parasites,we examined the effects of In vivo pretreatment with mAbs toICAM-1 (1A29) and LFA-1 (WT-1) on the protection against Infectionwith Listeria monocytogenesIn Fisher F344/N rats. Expressionof ICAM-1 and LFA-1 molecules on T cells In spleen, liver andperitoneal cavity of rats was down-regulated after i.p. administrationwith daily doses of 300 µg of either 1A29 or WT-1 for10 days. The survival rate of rats inoculated with viable Listeriawas significantly reduced byIn vivo pretreatment with 1A29 togetherwith WT-1 for 10 days but not by In vivo pretreatment with controlmAb. The numbers of bacteria In the spleen In rats pretreatedwith both 1A29 and WT-1 were significantly increased on day3 and day 6 after Infection with 1 x 10⁷ of viable Listeriacorresponding to 1/30 of LD₅₀ to normal rats. Thus, the resistanceagainst llsterial Infection was severely Impaired by combinationalpretreatment with mAbs In ICAM-1 and LFA-1. As shown In ourprevious report, the early appearance of CD3⁺TCRß^–T cells, presumably TCR cells, was evident In the peritonealcavity and liver of control rats at the early stage after llsterialInfection, while this was suppressed at this stage in rats pretreatedwith both 1A29 and WT1. These results suggest that the ICAM-1and LFA-1 adhesion pathway may be critically involved in protectlveroles of CD3⁺TCR_ß– T cells at the early stageof rat listeriosis.     

Synthesis and mutagenicity of 1-nitropyrene 4,5-oxide and 1-nitro-pyrene 9,10-oxide, microsomal metabolites of 1-nitropyrene

[4,5,9,10-³H]1-Nitropyrene was incubated with liver microsomesprepared from guinea pigs treated with Aroclor 1254 and theproducts were examined by h.p.l.c. The previously reported metabolites,1-nitropyrene trans-4,5-diliydrodiol, 1-nitropyrene trans-9,10-dihydrodiol,and 3-, 6-, and 8-hydroxy-1-nitropyrene were detected. In addition,h.p.l.c., nuclear magnetic resonance and mass spectral analysesindicated the presence of 1-nitropyrene 4,5-oxide and 1-nitro-pyrene9,10-oxide. The epoxide hydrase inhibitor, 1,2-epoxy-3,3,3-trichloropropane,decreased the concentration of the 4,5- and 9,10-dihydrodiolsin the microsomal incubations and increased the concentrationof their corresponding oxides. Reaction of 1-nitropyrene withm-chloroperoxybenzoic acid gave a mixture of 1-nitropyrene 4,5-oxideand 1-nitropyrene 9,10-oxide, which was separated by chromatography.The mutagenicity of the oxides was determined in Salmonellatyphimurium strains TA98, TA98NR, and TA98/1,8-DNP₆, both withand without exogenous activation by a rat liver homogenate fraction(S9). In the absence of S9, both oxides showed maximum activityin TA98, slightly decreased mutagenicity in the acetylase-deficientstrain TA98/1,8-DNP₆, and much reduced activity in the nitroreductase-deficientstrain, TA98NR. When assayed in the presence of S9, 1-nitropyrene4,5-oxide had maximum mutagenicity in TA98, and was 50 and 95%less mutagenic in TA98NR and TA98/1,8-DNP₆, respectively. 1-Nitropyrene9,10-oxide had a similar strain sensitivity, except that itstotal mutagenicity was lower. Since 1-nitropyrene is metabolizedby oxidative pathways in vivo, these K-region oxides may contributeto the toxicities elicited by this compound. ⁴To whom correspondence should be addressed     

The effect of biotransformation of 2,4-dinitrotoluene on its mutagenic potential

Because both oxidative and reductive metabolism of the hepatocarcinogen2,4-dinitrotoluene (2,4-DNT) can occur in vivo; we have examinedthe mutagenicity of compounds which can be formed from 2,4-DNTin an attempt to establish which metabolic pathways contributeto the formation of genotoxic products. A quantitative reversionassay using Salmonella typhimurium TA98 was used to evaluatethe mutagenicity of these compounds. 2,4-Dinitrobenzyl alcohol,2-amino-4-nitro-toluene and 2-nitroso-4-nitrotoluene were foundto be more mutagenic to S. typhimurium than is 2,4-DNT and didnot require metabolic activation by post-mitochondrial super-natantsof Aroclor-induced rat liver homogenates (S9) for their effect.2-Amino-4-nitrobenzoic acid was also mutagenic to S. typhimuriumTA98 in the absence of S9, but its mutagenicity was enhancedwhen S9 was included in the incubation mixture. 2,4-Diaminotoluenerequired S9 for demonstration of mutagenicity and was approximatelyas effective, on a molar basis, as 2,4-DNT in inducing reversionto histidine prototrophy. These results suggest that both oxidativeand reductive metabolism may be involved in production of mutagenicmetabolites of 2,4-DNT. ¹Present address to which correspondence should be addressed:Department of Pharmacology and Toxicology, University of MississippiMedical Center, 2500 N. State Street, Jackson, MS 39216, USA      

Effects of different antigenic microenvironments on the course of CD8+ T cell responses in vivo

The influence of microenvironment on the course of CD8⁺ T cellresponses in vivo was investigated by injecting H-2K^b-specificT cells from donor TCR transgenlc (TCR-Tg) mice into H-2K^b-tagmice. H-2K^b expression in recipients was either ubiquitous (CBKmice) or restricted to myeloid and erythroid cells (Kßmice). Donor T cells proliferated as extensively and acquiredsimilar surface phenotypes in spleen of both recipient types.Thus, neither the restricted pattern of H-2K^b expression northe significantly reduced level of H-2K^b expression by myeloldcells in Kß recipients affects the ability of thesplenic microenvironment to prime T cell proliferation in vivo.However, an unsustained burst of cytolytic activity was generatedrapidly in spleen of CBK recipients, whereas relatively littlecytolytic activity was generated in Kß spleen. Thisindicates that effector T cells were not generated efficientlyin spleen of Kß recipients even though extensive Tcell proliferation was taking place in this microenvironment.Furthermore, activated donor T cells dispersed rapidly throughoutprimary and secondary lymphoid organs of Kß recipients,whereas few T cells migrated from spleen in CBK recipients.Consequently, the course of CD8⁺ T cell responses and the anatomicaldistribution of activated T cells are profoundly influencedby the nature of the antigenlc microenvironment encounteredin vivo. We conclude that T cells rapidly proliferate and acquirenew tissue-homing characteristics but do not differentiate intocytolytic effector cells at the site of priming when they encountermyelold cells expressing low levels of antigen in vivo.     

Enhanced positive selection of a transgenic TCR by a restriction element that does not permit negative selection

Very little is known about the conformational properties ofthe MHC molecules that are able to signal positive selectionof a given TCR. To try to understand these parameters and todetermine whether these requirements are shared with interactionsduring negative selection and antigen recognition, we have studiedselection and antigen recognition of a transgenic TCR (specificfor lymphocytic choriomeningitls virus glycoprotein and H-2D^b)in the context of two D^b mutants, H-2^bm13 and H-2^bm14. The datashowed that the transgenic TCR was not positively selected bythe H-2^bm14 haplotype but, interestingly, enhanced positiveselection was seen in H-2^bm13 mice. The transgenic TCR couldnot be negatively selected In H-2^bm13animals persistently infectedwith the virus (neonatal virus carrier mice), nor could thetransgenic TCR be activated by H-2^bm13 infected cells in vivoor in vitro. These experiments show that although a TCR maybe selected by a mutant MHC molecule, the corresponding viralantigen cannot be recognized in the context of the mutant MHCmolecule, as Judged by both negative selection and T cell reactivityin vivo and in vitro. The ‘enhanced’ positive selectionoccurring in the context of D^bm13 suggests that a differentconformation of the MHC molecule is able to select the sameTCR and also that various TCR-ligand avidities may permit positiveselection.     

Evaluation of modified bacterial mutagenicity assays for the genotoxicity testing of mineral oils

A modified bacterial mutagenicity assay based on the Ames Salmonella/mammalianmicrosome test has been developed for application in the genotoxicitytesting of mineral oils. The assay uses washed microsomes fromrat liver in place of S9 fraction in order to increase the sensitivityof detection of genotoxicity. The modified assay was used totest a series of oils for which skin carcinogenicity bioassaydata in mice were available. Oils were tested as emulsions inwater using Tween 80 as a dispersant. A mutagenicity index foreach oil was obtained using non-linear regression analysis ofdata from the dose–response curve. The results showedan empirical correlation between increasing mutagenicity index,carcinogenicity and the polycyclic aromatic hydrocarbon contentof the oils. The washed-microsome assay was also compared withmodified Ames assays developed by Blackburn et al. (Cell Biol.Toxicol., 1, 40, 1984; Cell Biol. Toxicol., 2, 63, 1986) whichemployed increased levels of S9 (rat and hamster liver) to testdimethyl sulphoxide extracts of oils. The washed-microsome assaycan be used for the testing of whole oils rather than extractswhich are necessary for the modified Ames assay. It is recognisedthat the determinants of carcinogenic activity in vivo includepromoting activity which such assays are unable to detect. Nevertheless,such modified bacterial assays may be a useful prescreen sincegenotoxicity is recognised as a key initial step in carcinogenesis. ⁴To whom correspondence should be addressed at present address: Shell Research BV,Koninklijke/Shell Laboratorium Amsterdam, P.O Box 38000,1030 BN Amsterdam, The Netherlands     

Refinement of a T-lymphocyte cloning assay to quantify the in vivo thioguanine-resistant mutant frequency in humans

Cell cloning by limiting dilution in 96-well microtiter plateshas been employed to isolate colonies of human T-lymphocytesresistant to the purine analogue, 6-thioguanine (TG). Thesecolonies show stability of the TG-resistant (TG^r) phenotype,lack hypoxanthine guanine phosphoribosyl transferase (HPRT)activity and thus appear to be the result of in vivo somaticcell mutation events. In order to employ this T-lymphocyte cloningassay for quantitative determination of the in vivo TG^r mutantfrequency in humans, we have defined the optimal conditionsfor T-cell colony formation with both nonselected and TG-selectedcells. The parameters investigated include medium, serum, amountof the mitogen phytohaemagglutinin, amount of T-cell growthfactor (TCGF) and the number of irradiated feeder or accessorycells. Under the optimal conditions, the fraction of positivewells is proportional to the number of cells plated per wellwith both nonselection and TG selection conditions. T-cell cloningefficiencies therefore are independent of inoculum size. Therewas some evidence for a decline in TG^r mutant cell cloning atdensities >2 x 10⁴ cells per round-bottom well, possiblydue to metabolic cooperation between wild-type and mutant cells.The conditions defined in this study appear to provide a quantitativemeasurement of the in vivo TG^r mutant frequency in human T-lymphocytes.