Gene dosage mutants at adenine phosphoribosyltransferase locus induced by colcemid in Chinese hamster V79-AP4 cells

Pseudodiploid Chinese hamster V79-AP4 cells, functionally diploid at the adenine phosphoribosyltransferase (aprt)locus, were treated with colcemid, a well-known aneuploidizing agent, under various experimental conditions. Aneuploid and tetraploid cells and variants resistant to 10 g/ml of 2,6-diaminopurine (DAP), which selects for presumptive aprt^+/– heterozygotes in the untreated cells, were induced. Many of the induced variants were hypotetraploid with three (rather than four) chromosomes carrying the aprtgene. Dot-blot and Southern analysis of the DNA of these clones confirmed that they had three copies of the aprtgene. Their APRT specific enzymatic activity was 60–80% of that of wild-type V79-AP4. The results of these and other experiments suggest that in these variants resistance to DAP is due to an altered aprtgene dosage and point to a possible genetic effect of colcemid and other aneuploidizing agents in somatic mammalian cells.     

Deletion and hypermethylation of thymidine kinase gene in V79 Chinese hamster cells resistant to bromodeoxyuridine

Previous studies on V79 Chinese hamster cells have shown that bromodeoxyuridine (BrdU)-resistant variants deficient in thymidine kinase (TK) activity arise by a multistep process which is initiated by a random event and progresses gradually during serial culture in the presence of the drug. In order to determine the molecular basis for the loss of TK activity in these cells, the TK gene was isolated from a phage library of genomic V79 DNA, using a fragment of the human TK gene as a probe. One phage isolated contained the entire TK gene in a 15-kb insert, as demonstrated by the ability of the phage DNA to transform Ltk^}-mouse cells to the TK⁺ phenotype. Five fragments spanning the entire gene were then subcloned into the plasmid pUC12 for DNA methylation studies. With these probes it was shown by hybridization analysis that the copy number of the TK gene in V79 cells is about four times the copy number in CHO cells and Chinese hamster liver cells. Southern hybridization analysis of the DNA from first-stage variants partially resistant to BrdU indicated that partial resistance was accompanied by deletion of a number of copies of theha TK gene in V79 cells. However, the subsequent gradual transition to full BrdU resistance and full loss of TK activity was correlated with a gradual hypermethylation of sites in the 5 region of the TK gene, with no further change in gene copy number.     

Different mechanisms of reversion of HPRT-deficient V79 Chinese hamster cells

The revertibility of three spontaneous hypoxanthine phosphoribosyltransferase (HPRT)-deficient V79 cell lines has been determinedafter exposure to a number of alkylating agents. TGll and 19reverted at frequences ranging from 1 x 10"5 to 1 x 10"4 afterexposure to doses of ethyl-methane sulphonate (EMS) N-methyl-N-nitrosourea(MNU) and N-ethyl-N-nitrosourea (ENU) resulting in survivingfractions between 1.0 and 0.1. Reversion frequencies in TG15ranged from 10"7 to 5 x 10"6 over a similar dose range. Therelative efficiencies of different monofunctional alkylatingagents in causing reversion of TGll at equitoxic doses wereENU > EMS > N-ethyl-N-nitroso-guanidine MNU > N-methyl-N-nitrosoguanidine> methylmethane sulphonate. Revertant frequencies for allthree cell lines were maximal immediately after treatment anddeclined thereafter at a rate inversely proportional to dose.Such kinetics are explicable if reversion is due to miscodingopposite alkylated guanines. Reversion frequencies after N-butyl-N-nitrosoureaexposure were 100-fold lower than after MNU and kinetics ofexpression of revertant colonies differed. Frequencies werelow immediately after treatment, increased between 0 and 24h then remained at a plateau. Similar kinetics were observedafter chlorozotocin and bis-chloroethylnitrosourea exposure.This difference in expression kinetics suggests that reversionin this case is not the result of direct miscoding but of errorsin excision repair. TGll, 15 and 19 had low spontaneous mutantfrequencies which were either unaffected or only marginallyincreased by treatment with 5-azacytidine. The three HPRT" mutantsand revertants of TGll and 15 were subjected to Southern analysisusing a Chinese hamster HPRT cDNA as a probe. No differencesin restriction fragment patterns were observed and there wasno detectable amplification of HPRT sequences indicating thatthe HPRT" mutants and their revertants are the result of pointmutations. ²Present address: Baylor College of Medicine, Texas MedicalCenter, Houston, TX 77030, USA ³present address: Corporate Biosciences Laboratory. ICI plc,The Heath, Runcorn, Cheshire, UK      

Toxic effects of paracetamol and related structures in V79 Chinese hamster cells

Exposure of V79 Chinese hamster cells to non-cytotoxic concentrationsof paracetamol (4-hydroxyacetanilide, 4-HAA) increased sisterchromatid exchange (SCE) in the absence of an external activationsystem. Furthermore, a selective inhibition of DNA synthesiswas observed at low 4-HAA concentrations. The inhibition couldbe counteracted by the addition of ascorbate, indicating thatthe effect is caused by an oxidation product of 4-HAA. In attemptto clarify possible relationships between cytotoxicity, inhibitionof DNA synthesis and increased SCE, we studied the effect of4-HAA and some related structures on these parameters. The relativeposistion of the amino group and the hydroxyl group on the aromaticringappear to be important for the inhibition of DNA synthesis.Removal of either of the two groups, N-acetylation and/or alkylationof the aromatic ring or phenolic oxygen decreased the effectof the aromatic amine on DNA synthesis. A significant responseon SCE was observed with 4-aminophenol, 4-HAA, 2-HAA, 3, 5-dimethyl-4-HAA,3-HAA and 2, 6-dimethyl-4-HAA (none of the other compounds weretested). The increase in SCE frequency caused by 4-HAA and itsanalogs does not seem to be related to more general cytotoxiceffects. The relative potencies of the compounds for SCE inductionparalleled, for the most part, their effects on DNA synthesis.However, the induction of SCE and the inhibition of DNA synthesisdid not occur at comparable concentrations. Thus, the possibilitythat 4-HAA increases the frequency of SCE through some othermechanism cannot be excluded.     

Mutagenicity in V79 Chinese hamster cells of n-alkanals produced by lipid peroxidation

The mutagenicity for mammalian cells of five n-alkanals producedby lipid peroxidation was tested in V79 Chinese hamster lungcells either at the hypoxanthine-guanine phosphoribosyltransferaselocus as resistance to 6-thioguanine or at the Na/K ATPase locusas resistance to ouabain. The results show that propanal, butanal,pentanal and hexanal induced a dose-dependent increase in thefrequency over controls of both 6-thioguanine- and ouabain-resistantmutants at concentrations ranging from 3 to 30 mM. With nonanalthe same effects were observed with concentrations of 0.1–0.3mM.     

1,8-Dinitropyrene: comparative mutagenicity in Chinese hamster V79 and CHO cells

1,8-Dinitropyrene (1,8-DNP) was clearly mutagenic at the hprt locus in CHO cells, but not detectably mutagenic in V79 cells, following a 3-h treatment period. Preliminary data indicate that CHO, but not V79, cells have measurable levels of N-acetyltransferase activity, and this may contribute to the differential sensitivity of the two cell lines to the mutagenicity of 1,8-DNP.     

Mutation screening of bleomycin-induced V79 Chinese hamster hprt mutants using multiplex polymerase chain reaction.

We have shown by the filter hybridization technique that bleomycin (BLM) induces different types of mutations at the hprt gene locus of V79 Chinese hamster cells. DNA of mutants identified by Southern blots as partial deletions was subjected to further analysis using multiplex polymerase chain reaction (PCR) to localize the endpoints of the deletions over the hprt gene. The PCR analysis revealed that deletions occur in all parts of the hprt gene but are distributed non-randomly. Deletions occurred most frequently at the 3'-end of the hprt gene suggesting a possible existence of a hot spot for deletions in this region; exons 1, 2 and 3 appeared to be less affected by deletions. As PCR can detect microdeletions which are below the limit of resolution of Southern blot hybridization we analysed 25 HPRT- mutants with Southern wild-type pattern to distinguish between point mutations and small deletions. Of these HPRT- mutants, all except five showed PCR amplification products identical to that of V79 wild-type cells. These results are consistent with previous Southern analyses indicating that a large portion of BLM-induced HPRT- mutants are real point mutations. Five mutants, however, showed differences in fragment sizes of single PCR products or did not yield one single exon fragment and thus are probably the result of deletions which were not to be detected by Southern analyses.     

Effects of antioxidants on V79 Chinese hamster cells treated with ferric nitrilotriacetate

The cytotoxic effects of ferric nitrilotriacetate (Fe-NTA) have been considered to be caused by free radicals produced by the drug. The present study was carried out to determine whether or not cytotoxic effects of Fe-NTA on cell growth and lipoperoxide formation of Chinese hamster cells were reduced by antioxidants. Using a spin trapping technique, we found that hydroxyl radical formation in the cells increased in the presence of Fe-NTA. Antioxidants, with the exception of superoxide dismutase, slightly inhibited production of the hydroxyl radical. Mannitol significantly reduced lipoperoxide formation, but other antioxidants did not. However, the growth inhibitory effects of Fe-NTA were not attenuated by these antioxidants. These results indicated that the cytotoxic effects of Fe-NTA may be mostly due to unknown factors other than oxygen free radicals.     

Gene amplification and gene deletion are induced at different rates in V79/AP4 Chinese hamster cells

The effect of mitomycin C (MMC) treatment on gene amplificationand gene deletion induction in a V79/AP4 Chinese hamster cellline was investigated. Spontaneous and induced cellular variantsresistant to N-phosphonacetyl-L-aspartate (PALA), which selectsfor carbamyl-P-synthetase, aspartate transcarbamylase and dihydroorotase(CAD) gene amplification events, and to intermediate concentrationsof 2,6-diaminopurine (DAP), which selects for adenine phosphoribosyltransferase (aprt) gene deletion events, were isolated. Themolecular analysis of spontaneous and induced PALA- and DAP-resistantclones showed that while in the former the CAD gene was amplified,in the latter both mutations and deletions occurred at the aprtgene. These results indicate that MMC favored the rise of geneamplification rather than gene deletion events, and suggestthat aberrant DNA replication processes were responsible forthe observed gene amplification. ¹To whom correspondence should be addressed     

Mutagenicity of N-substituted phenanthrene 9,10-imines in Salmonella typhimurium and Chinese hamster V79 cells

We previously showed that some (nonsubstituted) aziridines derived from polycyclic aromatic hydrocarbons (arene imines) elicit various mutagenic and genotoxic effects in bacteria and mammalian cells and that these arene imines are active at much lower concentrations than the corresponding epoxide analogues. In the present study, N-substituted derivatives of phenanthrene 9,10-imine were investigated. All 10 derivatives studied showed direct mutagenicity in Salmonella typhimurium TA100. Some of the compounds additionally exhibited weak effects in the strains TA98 and TA1537. Most N-substituted derivatives were weaker mutagens than unsubstituted phenanthrene 9,10-imine but stronger mutagens than phenanthrene 9,10-oxide. Bulky substituents reduced the mutagenicity more than did small substituents. In addition, the derivatives with electron-withdrawing substituents (with the exception of N-chlorophenanthrene 9,10-imine) were weaker mutagens than those with electron-donating substituents. Phenanthrene 9,10-imine and five N-substituted derivatives were investigated to determine whether they induce gene mutations at the hgprt locus in V79 cells. Four compounds, including the parent aziridine, were positive in the V79 test. The other two compounds were negative. The mutagenic potencies in the V79 cell system did not correlate well with those obtained with the Salmonella system. Overall, the study shows that in addition to unsubstituted arene imines, N-substituted derivatives are mutagenic. This finding is of interest, as metabolic pathways leading from aromatic compounds to N-substituted arene imines are conceivable.     

DNA-PK inhibitor wortmannin enhances DNA damage induced by bleomycin in V79 Chinese hamster cells

The fungal metabolite wortmannin (WM) is a potent and irreversible inhibitor of the enzyme DNA-dependent protein kinase (DNA-PK), a nuclear serine-threonine kinase, member of the phosphaditylinositol-3 kinase related kinase family. WM has been used in the last few years as a promising radiosensitizer mainly throughout cell survival experiments. However, few studies have addressed the role of DNA-PK inhibition in the repair of DNA lesions generated by antitumor agents. Bleomycin (BLM) is an antitumor agent used in the treatment of various neoplasia with a unique genotoxicity profile that mimics the ionizing radiation effects. In this study, we evaluated the effect of different concentrations of WM on the DNA damage induced by BLM. The cytokinesis-block micronucleus assay (CBMN) in V79 Chinese hamster cells was used as the end-point. WM significantly increased the frequency of micronucleated cells (%MNBN) by about 2.2-fold, the number of micronuclei per binucleated cell (MN/BN) by about 2.4-fold, and also changed the pattern of the distribution of micronuclei induced by BLM. The frequency of micronucleated cells with 2 MN per cell and with > or = 3 MN per cell increased, whereas the frequency of micronucleated cells with 1 MN per cell decreased. WM was not genotoxic but decreased cell proliferation as assessed by the frequency of binucleated cells. Our results show that WM clearly enhances the efficacy of BLM in terms of DNA damage inflicted and therefore reinforces its use as a chemosensitizer.     

In vitro micronucleus screening of pharmaceutical candidates by flow cytometry in Chinese hamster V79 cells

We previously reported a high concordance of in vitro micronucleus (MNvit) results obtained by flow cytometry to the known cytogenetic activity often commercially available compounds mentioned as validation compounds in an early draft of the OECD MNvit TG487 [Bryce et al., 2010; Organization for Economic Co-operation and Development(OECD), 2007]. The current study investigated this method in Chinese hamster V79 cells with pharmaceutical compounds of unknown genotoxic potential. Twenty-five compounds from several therapeutic areas such as oncology, neuroscience and immunological research were tested in the flow cytometry assay, and for comparison using the cytokinesis-block microscopy assay. Five of these 25 compounds were considered positive for micronucleus induction by the microscopy assessment. In all cases, the results from the flow cytometry assess ment matched the results of the microscopy assay. Thus, flow cytometry is a viable method for assessing the aneugenic/clastogenic potential of pharmaceutical drug candidates. The flow method offered several advantages over traditional microscopy. For instance, the ratio of micronuclei (MN) to 10,000 nuclei was evaluated in less than 2 min vs.15 min to manually assess 600 binucleate cells. Evaluation by flow cytometry can be automated,freeing resources and eliminating scorer fatigue.The assay may also provide for mechanistic understanding of MN formation based on size and the ratio of nuclei with sub-2N DNA content, allowing for discrimination between aneugenic and clastogenic compounds.     

Induction of genetic instability and chromosomal instability by nickel sulfate in V79 Chinese hamster cells

Nickel compounds are known to be carcinogenic to humans and show genotoxicity, including the ability to induce chromosome aberrations and neoplastic transformation in vitro. The mutagenicity of nickel compounds is, however, equivocal and the mechanisms of carcinogenesis are still not clear. In this study, the possibility that nickel compounds induce genetic or chromosomal instability was examined, because recent studies in cancer research show that these conditions are critically involved in carcinogenesis. V79 Chinese hamster cells were treated with 320 microM nickel sulfate for 24 h at low cell density (100 cells/100 mm diameter dish) and clones derived from single cells surviving Ni treatment were isolated. When cells grew up to 23-25 population doublings post-treatment, mutation frequency at the HPRT locus and the chromosome aberration frequency of each clone were examined. Five out of 37 clones (13.5%) derived from Ni-treated cells showed a remarkably increased frequency of HPRT mutations (>or=1 x 10(-4)), while only one out of 37 control clones (2.7%) showed this high mutation rate. In addition, 17 out of 37 clones (45.9%) from Ni-treated cells showed structural chromosomal aberrations in 10% or more of cells (up to 45.5%), while only three out of 31 control clones (9.7%) showed this high aberration rate. Out of 37 clones derived from Ni-treated cells, eight (21.6%) and 11 (29.7%) clones showed an increased frequency (>or=5%) of aneuploid and polyploid cells, respectively, while only a few control clones showed such an increase in aneuploid and polyploid cells. These results indicate that nickel sulfate can induce genetic and chromosomal instability in V79 cells.     

The Chinese hamster V79 cell mutant V-H4 is phenotypically like Fanconi anemia cells

It has been shown by genetic complementation analysis that a mitomycin C-sensitive mutant (V-H4) of Chinese hamster V79 cells is the first rodent equivalent of Fanconi anemia (FA) group A. The V-H4 mutant shows many typical characteristics of cells derived from FA patients. V-H4 cells exhibit increased sensitivity towards cross-linking agents as MMC (30-fold), cis-DDP (10-fold), DEB (10-fold), and PUVA (1.6-fold), but an only slightly increased sensitivity to monofunctional alkylating agents (EMS and MMS) and actinomycin D. V-H4 cells are also moderately sensitive to adriamycin (1.6-fold), and not sensitive to H₂O₂. The levels of chromosomal aberrations induced by MMC and cis-DDP treatment are higher (4- to 6-fold) in V-H4 cells than in the wild-type V79 cells. Genetic complementation analysis with other Chinese hamster mutants hypersensitive to MMC (irs1, irs1SF, UV20 and UV41) indicates clearly that V-H4 belongs to a different, new complementation group. This unique mutant is very stable and can serve as a vehicle to isolate the complementing FA-A gene from normal human DNA.     

Arsenic[III] and heavy metal ions induce intrachromosomal homologous recombination in the hprt gene of V79 Chinese hamster cells

In the present study the carcinogenic metal ions Cd[II], Co[II], Cr[VI], Ni[II], and Pb[II], as well as As[III], were examined for their ability to induce intrachromosomal homologous and nonhomologous recombination in the hprt gene of two V79 Chinese hamster cell lines, SPD8 and Sp5, respectively. With the exception of Pb[II], all of these ions enhanced homologous recombination, the order of potency being Cr>Cd>As>Co>Ni. In contrast, Cr[VI] was the only ion to enhance recombination of the nonhomologous type. In order to obtain additional information on the mechanism of recombination in the SPD8 cell line, individual clones exhibiting metal-induced recombination were isolated, and the sequence of their hprt gene determined. These findings confirmed that all recombinogenic events in this cell line were of the homologous type, involving predominantly a chromatid exchange mechanism. The mechanisms underlying the recombination induced by these ions are discussed in relationship to their genotoxicity, as well as to DNA repair and replication. Induced recombination may constitute a novel mechanism for induction of neoplastic disease.     

Use of catalytic topoisomerase II inhibitors to probe mechanisms of chemical-induced clastogenicity in Chinese hamster V79 cells

Determination of the clastogenic potential of new chemical entities, particularly pharmaceuticals, is an important part of the overall safety assessment of such drugs. It is appreciated that clastogenicity can arise from perturbation of many different cellular processes distinct from direct DNA/drug interactions. One such alternative clastogenic process is inhibition of DNA topoisomerase II, during which process the topoisomerase/DNA/drug ternary complex forms stable DNA double-strand breaks (cleavable complex), which become templates for recombinational, mutagenic, and chromosomal fragmentation events. Without extensive experimentation, it is generally not possible to distinguish clastogenicity arising from direct drug/DNA interaction from that arising from inhibition of topoisomerase II. In the present investigation, we demonstrate that specific catalytic inhibitors of DNA topoisomerase II reduce the clastogenicity of topoisomerase poisons but not that arising via non-topoisomerase-dependent mechanisms. In particular, it is shown that catalytic topoisomerase II inhibitors such as chloroquine, sodium azide, and A-74932, as well as certain intercalating agents such as 9-aminoacridine and ethidium bromide, strongly antagonize the formation of micronuclei induced by the DNA gyrase inhibitor clinafloxacin and the antitumor topoisomerase II poison etoposide. These catalytic inhibitors are also shown to antagonize the clastogenicity of experimental compounds and novel pharmaceuticals presumed to be DNA intercalating agents by virtue of their response in a cell-based bleomycin amplification assay. We extend our previous hypothesis, suggesting that the clastogenicity of some nonstructurally alerting drugs may be due to an as yet unappreciated propensity for DNA intercalation. It is further proposed that intercalation-dependent inhibition of DNA topoisomerase II may be responsible for this clastogenicity and that this may be detected in intact mammalian cells with the use of catalytic topoisomerase inhibitors.     

Characterization of the genotoxic potential of formaldehyde in V79 cells

Formaldehyde (FA) is known to be genotoxic and mutagenic in proliferating mammalian cells in vitro. The present study was performed to further characterize its genotoxic potential in the V79 Chinese hamster cell line. The induction of DNA strand breaks and DNA-protein cross-links (DPXs) was measured by the comet assay in relationship to the induction of sister chromatid exchanges (SCEs) and micronuclei (MN). Induction of DNA strand breaks was found neither with the standard protocol of the alkaline comet assay nor with modifications using extended electrophoresis times or proteinase K. The concentration-effect relationship for the genotoxic effects was characterized by fitting different curves to the data. A two-phase regression model fitted best in comparison with a linear or a quadratic model and indicated practical thresholds for the induction of SCE and MN. For the induction of DPX as measured by the comet assay, neither a linear concentration-response relationship nor any of the tested models fitted well to the data. Three repeated treatments with genotoxic concentrations of FA with a 3-h interval led to enhanced levels of DPX and MN while the same treatments with a 24-h interval did not enhance FA genotoxicity but suggested adaptive protection against the DNA-damaging action of FA.     

Nickel potentiates the genotoxic effect of benzo[a]pyrene in Chinese hamster lung V79 cells

The modulating effect of acute exposure to NiCl2 on the induction of chromosome aberrations by a model carcinogen, benzo[a]pyrene (B[a]P), was examined in Chinese hamster V79 lung cells. At concentrations up to 20 microg/ml (84.2 microM), NiCl2 did not significantly increase the frequency of chromosome aberrations in V79 cells when the cells were exposed concomitantly to 0.5 microg/ml B[a]P. Addition of the S15 liver microsomal fraction together with the B[a]P did not alter the results. Addition of NiCl2 2 hr before treatment of cells with 0.5 microg/ml B[a]P also did not result in a significant elevation of the frequency of chromosome aberrations, even at NiCl2 concentrations as high as 20 microg/ml. Contrasting sharply with these findings, when V79 cells were treated with NiCl2 immediately after B[a]P exposure, a significant increase in the frequency of chromosome damage was observed at NiCl2 concentrations as low as 5 microg/ml (21.1 microM). NiCl2-mediated enhancement of chromosome damage was also observed when V79 cells were exposed to the reactive B[a]P intermediate, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BPDE). In the BPDE-treated cells, the level of NiCl2-mediated enhancement was similar to that observed with the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA, 100 ng/ml). These results are consistent with the view that the effect of nickel (II) on B[a]P-induced genetic damage is dependent on the relative times of exposure to Ni2+ and B[a]P. NiCl2 did not enhance the frequency of chromosome aberrations induced by Chromium (VI), regardless of the order of addition of the chemicals to the V79 cells. These results suggest that nickel may act as a promoter of chemically-induced genetic damage through induction of error-prone repair.     

Hydrogen peroxide-induced activation of SAPK/JNK regulated by phosphatidylinositol 3-kinase in Chinese hamster V79 cells

To clarify activation mechanisms of stress-activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) during oxidative stress, the roles of phosphatidylinositol 3-kinase (PI 3-kinase), concentration of intracellular calcium ([Ca2+]i), and cyclic AMP-dependent kinase (PKA) in hydrogen peroxide (H2O2)-induced SAPK/JNK activation were examined in Chinese hamster V79 cells. SAPK/JNK was dose-dependently activated after H2O2 treatment (from 10 microM to 1 mM), and a PI 3-kinase inhibitor (wortmaninn), intracellular calcium chelator (BAPTA-AM), and PKA activator (dibutyl cyclic AMP and forskolin) inhibited this activation. An increase in [Ca2+], was observed after treatment with H2O2. Immunoprecipitation revealed that a PI 3-kinase regulatory subunit, p85alpha, was associated with insulin receptor substance 1 (IRS-1) phosphorylated by H2O2 treatment. Furthermore, the formation of this complex of p85alpha and phospho-IRS-1 was abolished by the presence of BAPTA-AM but not forskolin. These results indicated that the PI 3-kinase activated through phosphorylation of IRS-1 upstream of SAPK/JNK after H2O2 treatment of V79 cells and that [Ca2+]i was a regulation factor for phosphorylation of IRS-1.     

Influence of hyperthermia, pH and culturing conditions on the electrical parameters of Chinese hamster V79 cells

Conductivity measurements in the frequency range 10 kHz-100 MHz have been performed on Chinese hamster fibroblasts V79 exposed to hyperthermia in different experimental conditions. The membrane electrical conductivity is decreased when cells are heated for 1 h at 44 degrees C or 3 h at 43 degrees C at pH 7.25. This effect has not been observed on cell samples following heating for 1 h at 43 degrees C at pH 7.25. However, if the pH of the culture medium is adjusted to 6.50 or 6.0 before hyperthermic exposure, a substantial decrease of membrane conductivity is observed. In cells maintained in partially spent medium before heating, a 3 h treatment at 43 degrees C at pH 7.25 does not produce any effect on membrane conductivity. The results of all these experiments seem to indicate that some alteration of the energy-driven mechanisms involved in active transport across the plasma membranes may be responsible for the observed effects.