Mouse Leukemia L1210 Cells Research Articles

EARLY PROGRESSION FROM DIMETHYL SULFOXIDE-INDUCED G0/G1 ARREST IN L1210 CELLS

Recently, dimethyl sulfoxide (DMSO) has been used as a convenient cryoprotectant for stem cells in stem cell transplantation using allogenic peripheral blood or umbilical cord blood. As the stem cells have a multipotency, clarification of the extent of cell proliferation after transplantation is difficult. In the present study, DMSO gradually induced G0/G1 arrest in mouse leukemia L1210 cells with good cell viability. After removal of DMSO, the cells proliferated appropriately, resulting in expression of the DNA-synthesizing enzymes thymidylate synthase and thymidine kinase within 6h, and the cells entering into S phase within 12h. The sequence was followed by the marked activation of both enzymes within 24h and the increase of bromodeoxyuridine (BrdU) immunoreactive (S phase) cells with rapid cell proliferation within 36h. In conclusion, mouse leukemia L1210 cells, which were treated with 1.5% DMSO for 96h, tolerated the treatment and reversed the cell cycle arrest within 36h.

Overexpression of protein disulfide isomerase-like protein in a mouse leukemia L1210 cell line selected for resistance to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, a ribonucleotide reductase inhibitor.

Overexpression of protein disulfide isomerase-like protein in a mouse leukemia L1210 cell line selected for resistance to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, a ribonucleotide reductase inhibitor.

Synthesis and biological studies of novel nucleoside phosphoramidate prodrugs.

A novel approach to the intracellular delivery of nucleotides using phosphoramidate-based prodrugs is described. Specifically, we have developed phosphoramidate prodrugs of the anticancer nucleotide 5-fluoro-2'-deoxyuridine-5'monophosphate (FdUMP). These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation liberating phosphoramidate anion, which undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quantitatively. In vitro evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs 2a and 3b against L1210 mouse leukemia cells show potent inhibition of cell growth (IC(50) 0.5-3 nM). Cell-based thymidylate synthase inhibition studies show that, in contrast to FUdR, the nitrofuran compound 2a is of comparable potency in wild type vs thymidine kinase deficient LM cells. This result indicates that the activation of this novel prodrug occurs via the proposed mechanism of intracellular delivery. However, naphthoquinone 3b has an IC(50) value for thymidylate synthase inhibition that is comparable to FUdR in thymidine kinase deficient cells. Further studies revealed that 3b rapidly decomposes to the nucleotide in cell culture medium, suggesting that the naphthoquinone analogue is not sufficiently stable to function as a nucleotide prodrug.

Structural characterization and cytostatic activity of chlorobischolylglycinatogold(III).

Based on the ability of bile acids for vectorializing the cytostatic activity of other agents, we have designed and synthesized a new bile acid cholylglycinato Au(III) complex, named Bamet-A1. It has been characterized by means of EA (elemental analysis), FT-IR, NMR, FAB-MS (fast atom bombardment-mass spectrometry) and Vis-UV techniques. This characterization allowed us to propose a structure of the type [Au CG(O) CG(N,O) Cl] for the neutral complex, which has the composition C522H84N2O12AuCl and is very soluble in water, methanol, ethanol and DMSO (dimethylsulfoxide). The study in aqueous solution suggested a redox process for its transformation, which is accompanied by the appearance of colloidal gold phase. The behavior in 4 mM NaCl water (in order to mimic the cytoplasmatic fluid) was similar to that observed in water, while in a 150 mM NaCl (similar to extracellular fluid and serum), the apparition of a dark blue precipitate was observed. This complex displays fluorescence, which does not change when incubated with DNA obtained from E. coli. Bamet-A1 was found to inhibit the growth of a variety of cell lines. The cytostatic effect was mild against human hepatoma HepG2, mouse hepatoma Hepa 1-6, rat hepatoma McA RH-7777 and human colon adenocarcinoma LS-174T, and stronger against mouse sarcoma S180-II and mouse leukemia L-1210 cells. The appearance of colloidal Au during the process of hydrolysis under physiological conditions may explains the low cytostatic activity.

The ribonucleotide reductase inhibitor trimidox induces c-myc and apoptosis of human ovarian carcinoma cells

Trimidox (3,4,5-trihydroxybenzohydroxamidoxime), a recently synthesized inhibitor of ribonucleotide reductase (RR), was shown to exert anti-proliferative activities in HL-60 and K562 human leukemia cell lines and to prolong the life span of mice inoculated with L1210 mouse leukemia cells. Here we test whether trimidox also exhibits anti-neoplastic properties in ovarian carcinoma cells. Since the mode of action of trimidox on cell fate has not been investigated so far, we addressed this unresolved item and find that this polyhydroxybenzoic acid derivative induces apoptosis of N.1 human ovarian carcinoma cells when tested in growth factor deprived medium. Utilizing an improved analysis, based on Hoechst 33258 / propidium iodide double staining, apoptosis is quantified and discriminated from necrosis. Trimidox induces c-myc expression, which is indispensible for apoptosis of N.1 cells, and expression of plasminogen activator / urokinase type (upa), which supports the apoptotic process under more physiological conditions. Surprisingly, trimidox does not block dNTP synthesis in N.1 cells at the concentrations tested and, therefore, trimidox induces apoptosis independent of RR-inhibition. Like TNFα or benzamide riboside, which are also inducers of apoptosis of N.1 cells, trimidox also down-regulates the G1 cell cycle phosphatase cdc25A, whereas cyclin D1 becomes up-regulated. This report shows that trimidox destroys human ovarian carcinoma cells by inducing them to undergo apoptosis as well as corroborating previous investigations which demonstrated that apoptosis of these cells depends on c-myc over-expression when survival factors are withdrawn.

Synthesis and biological activity of novel 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs.

A series of novel haloethyl and piperidyl phosphoramidate FdUMP prodrug analogues has been synthesized, and the growth inhibitory activity of these compounds has been evaluated against L1210 mouse leukemia cells. All compounds exhibited potent inhibition of L1210 cell proliferation with IC(50) values in the nanomolar range. Growth inhibition was reversed by the addition of 5 microM thymidine, suggesting a mechanism of action involving the intracellular release of FdUMP. (31)P NMR studies carried out on model haloethyl phosphoramidates confirm the release of nucleotide via cyclization of the phosphoramidate anion to the aziridinium ion intermediate followed by hydrolysis of the P-N bond. The data suggests that <50% of the prodrug is converted to FdUMP intracellularly by this pathway. Piperidyl phosphoramidate analogues are also converted to nucleotide intracellularly, presumably by the action of an endogenous phosphoramidase.

Altered sensitivity of deoxyadenosine-resistant mouse leukemia L1210 cells to apoptosis induced by 7-hydroxystaurosporine.

The deoxyadenosine-resistant mouse leukemia L1210 cell line (Y8) has previously been shown to be more sensitive to apoptosis induced by DNA damaging agents and by protein synthesis inhibitors than the parental wild-type L1210 (WT) cells. These responses occur independently of p53 as both cell lines lack wild-type p53 function. Recent evidence suggests that a serine/threonine kinase is involved in the divergent cellular responses of the WT and Y8 cells. In the present study, the effects of 7-hydroxystaurosporine (UCN-01), a relatively specific serine/threonine kinase inhibitor, were examined in the WT and Y8 cells. Both cell lines were equally sensitive to the growth inhibitory effects of UCN-01. However, the Y8 cells accumulated in G0/G1 and became apoptotic. Apoptosis induced by UCN-01 in the Y8 cells was mediated by a caspase-3-like activity which could be partially blocked by Ac-DEVD-CHO, a caspase-3 inhibitor. UCN-01 did not alter the phosphorylation status of cdc2 nor cyclin B1 and cdc2 protein levels in either cell line.

Spermine Oxidation Leads to Necrosis with Plasma Membrane Phosphatidylserine Redistribution in Mouse Leukemia Cells

Oxidation by copper/quinone-containing serum amine oxidases (SAO) is a well-known cause of polyamine cytotoxicity. Spermine oxidation exerts potent immunosuppressive effects in animal cells, but the cell death mechanism involved remains unclear. We compared biochemical and morphological parameters of SAO-mediated cell death in L1210 mouse leukemia cells with normal or amplified ornithine decarboxylase gene expression with those observed during apoptosis induced by deregulated polyamine uptake or by okadaic acid. None of the characteristic features of apoptotic cell death (e.g., chromatin condensation, nuclear fragmentation, internucleosomal DNA cleavage, poly(ADP-ribose) polymerase cleavage) were observed during spermine oxidation-mediated cell death, which was clearly necrotic by morphological criteria. Inhibition of a wide spectrum of caspases did not prevent SAO-dependent cell death, whereas N-acetylcysteine completely abolished the cytotoxic effects of spermine oxidation. Catalase only delayed spermine oxidation-induced cell death without affecting its modality or preventing depletion of intracellular glutathione, suggesting that both H2O2 and aminoaldehydes generated by SAO-mediated spermine oxidation contribute to SAO-induced necrosis. Interestingly, redistribution of phosphatidylserine to the outer leaflet of the plasma membrane, usually a diagnostic feature of apoptosis, preceded necrotic cytolysis triggered by spermine oxidation. Thus, L1210 cell death caused by SAO-mediated spermine oxidation has all the attributes of primary necrosis, but is also accompanied by loss of phospholipid asymmetry, indicating that the latter phenomenon may not be unique to apoptosis. Phosphatidylserine exposure, a potent engulfment signal for phagocytes, might contribute to the immunosuppressive effects of plasma polyamines through a controlled and rapid necrotic process involving SAO.

Phenotypic changes in mouse leukemia L1210 cells with alterations in the effector-binding subunit of ribonucleotide reductase.

Phenotypic changes in mouse leukemia L1210 cells with alterations in the effector-binding subunit of ribonucleotide reductase.

Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cells.

Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3'-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3'-UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3'-UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating approximately 65-90 kDa native reduced folate carrier and a approximately 40-45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-[3H]MTX and NHS-[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed.

Determinants of the apoptotic response to lysosomal photodamage.

Studies with mouse leukemia L1210 cells revealed that selective lysosomal photodamage caused by any of three photosensitizing agents was followed by a gradual loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol, increased DEVDase activity (a measure of levels of caspase-3) and a limited apoptotic response. Similar effects were observed in the murine hepatoma 1c1c7 cell line. Immunofluorescence techniques employing 1c1c7 cells demonstrated the immediate release of the lysosomal enzyme cathepsin B following lysosomal photodamage. These studies suggest that the cytotoxic effects of lysosomal photodamage are initiated by released lysosomal proteases that either directly and/or indirectly activate caspases as a consequence of the induction of mitochondrial damage.

2- or 6-(1-azidoalkyl)-5,8-dimethoxy-1,4-naphthoquinone: synthesis, evaluation of cytotoxic activity, antitumor activity and inhibitory effect on DNA topoisomerase-I.

6-(1-azidoalkyl)-DMNQ derivatives compared to 2-(1-azidoalkyl)-DMNQ isomers, exhibited higher cytotoxic activity against L1210 mouse leukemia cells and stronger inhibition of DNA topoisomerase-I (TOPO-I), suggesting involvement of steric hindrance. However, similar antitumor activity against mice bearing S-180 cell was shown by 2- and 6-(1-azidoalkyl)-DMNQ derivatives.

Mouse leukemia L1210 cells selected for resistance to the ribonucleotide reductase inhibitor, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone show altered response to DNA damaging agents.

An L1210 cell line (MQ-580) selected for resistance to the ribonucleotide reductase inhibitor, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ), had been previously shown to have altered properties related to the non-heme iron subunit of ribonucleotide reductase (RR). In addition, the MQ-580 cells had other metabolic alterations that were consistent with multidrug resistance. Neither the wild-type (WT) nor the MQ-580 cells showed a G0/G1 block in response to X-irradiation. Since the MQ-580 cells also showed resistance to adriamycin (ADR), the WT cells were more sensitive to the effects of ADR than the MQ-580 cells. However, when the MQ-580 cells were treated with the combination of ADR plus verapamil, the MQ-580 cells showed cellular responses that included cell cycle block in G2/M and increased apoptosis. The WT cells, in response to the combination of ADR plus verapamil, blocked in the S-phase of the cell cycle with an increased necrotic cell population in comparison to treatment with ADR alone. While MAIQ could be shown to cause an apoptotic response in the WT and MQ-580 cells, the concentrations of MAIQ required to induce apoptosis were 20- to 40-times the IC50 value. However, even though the MQ-580 cells were 8-fold more resistant to MAIQ than the WT cells, the MQ-580 cells were more sensitive to MAIQ-induced apoptosis. These data show that alterations at the RR site lead to phenotypic expressions not overtly related to RR. However, because of the critical role that RR plays in cell division and DNA repair, the changes observed with respect to cell cycle and apoptosis, may in fact, be a direct consequence of the alteration at RR.

A Tetranuclear Platinum Compound Designed to Overcome Cisplatin Resistance

The synthesis and characterisation of the first generation of a poly(propyleneimine) dendrimer DAB(PA)4, substituted with four trans-diamminechloroplatinum moieties is reported. The compound DAB(PA-tPt-Cl)4 was designed to overcome two problems often associated with cisplatin resistance in cancer cells: (i) deactivation of the platinum species by intracellular thiolates and (ii) improved repair of crosslinks with DNA. The four-armed molecule can be expected to form crosslinks with DNA that are very different from the adducts formed by cisplatin. Also, the tetranuclear compound has four leaving groups, while cisplatin has only two. Therefore, DAB(PA-tPt-Cl)4 would be less susceptible towards inactivation by reaction with intracellular thiolates. A reaction with an excess of the model nucleobase guanosine 5′-monophosphate (GMP) confirmed that the tetranuclear compound is capable of binding a maximum of four nucleobases. Therefore, the inactivation of one or two arms would still leave the molecule with enough reactivity to form crosslinks with DNA. Cytotoxicity tests were performed on two mouse leukemia L1210 cell lines, both sensitive and resistant towards cisplatin, and in seven human tumor cell lines. In all cell lines, the tetranuclear compound showed a low cytotoxicity. It is suggested that the low activity is related to the structure of the compound. Probably the high charge (+6) at physiological pH and its branched structure hamper the molecule in crossing the cell membranes.

Nucleosides and nucleotides. 185. Synthesis and biological activities of 4'alpha-C-branched-chain sugar pyrimidine nucleosides.

A series of 4'alpha-C-branched-chain pyrimidine nucleosides was synthesized from 2'-deoxycytidine or uridine. In the 2'-deoxycytidine series, the substituent at the 4'alpha-position affected cytotoxicity against L1210 mouse leukemic cells in vitro in the order Me (23) > CN (22) > C(symbol)CH (21) > CH=CH(2) (19) > Et (24) > CH=CHCl (20). However, uridine and cytidine derivatives with ethynyl and cyano groups at the 4'alpha-position did not show any cytotoxicity. The antiviral activities of these nucleosides against HSV-1, HSV-2, and HIV-1 in vitro were also examined. Compounds 22 and 23 showed antiviral activities against HSV-1 and HSV-2 without showing significant toxicity to the host cells (MRC-5 cells). Although almost all of the nucleosides showed anti-HIV-1 activities, they were also cytotoxic to the host cells (MT-4).

Effect of doxorubicin on wild-type and deoxyadenosine-resistant mouse leukemia L1210 cells.

Wild-type (WT) mouse leukemia L1210 cells express steady-state levels of the mRNA and protein for p53. However, the p53 expressed by the wild-type cells is a mutant form of p53. A deoxyadenosine-resistant L1210 cell line (Y8) derived from the parental WT L1210 cells does not maintain constitutive levels of p53 mRNA or protein. Upon DNA damage, induced by doxorubicin, neither the WT nor the Y8 cells block in G0/G1; the cells block in G2/M. However, treatment of the Y8 cells with doxorubicin results in a much greater fraction of cells becoming apoptotic compared to the WT cells. Doxorubicin treatment resulted in the induction of p53 mRNA in the WT cells, but not the Y8 cells. WAF1, c-myc and Bax mRNAs were also induced by doxorubicin in the WT cells but not in the Y8 cells. The constitutive levels of WAF1 and Gadd45, unexpectedly seen in the p53-deficient Y8 cells, decreased following doxorubicin treatment. The comparison of the effects of DNA damage, as measured by mRNA levels, induced by X-irradiation or doxorubicin were found to vary between the WT and Y8 cells and for the particular mRNA studied. The effect of doxorubicin or X-irradiation on the cell cycle could be overridden in the WT cells by caffeine. Comparisons of DNA damage induced by doxorubicin or X-irradiation show that although the Y8 cells are more sensitive to these damaging agents than the WT cells, the effects on gene expressions are not identical.

Synthesis of 2,8-disubstituted imidazo[1,5-a]pyrimidines with potent antitumor activity.

Seventeen 1,2,3,4-tetrahydroimidazo[1,5-a]pyrimidine derivatives bearing electron-withdrawing substituents were designed and synthesized by novel ring closure as potential antitumor agents. They were screened for their activities against mouse leukemia L1210 and human oral epidermoid carcinoma KB cell lines, and relationships of structure and antitumor activity in vitro are discussed. It was found that 8-thiocarbamoyl-1,2,3,4-tetrahydroimidazo[1, 5-a]pyrimidin-2(1H)-thione (8c) exhibited activity comparable to that of 5-fluorouracil against both L1210 and KB cells. The existence of both 2-thioxo and 8-substituent with a thioxo group in the molecule is crucial for the cytotoxicity against L1210 and KB cells. A novel procedure for introduction of a double bond between C-3 and C-4 in 8c was developed. Introduction of the 3,4-double bond increased the activity against L1210, but against KB cells the activity decreased by 4-fold. Cytotoxicity of compounds 8c and 8-thiocarbamoyl-1,2-dihydroimidazo[1,5-a]pyrimidin-2(1H)-thione (11c) against human solid tumor and leukemia cell lines was further evaluated. The saturation of the 3,4-double bond led to a significant increase in cytotoxicity against tumor cell lines tested.

KATP channel blocking actions of quaternary ions play no role in their antiproliferative action on mouse leukaemia and rat vascular smooth muscle cells in vitro.

1. The aim of the present study was to investigate the possibility that, in the two cell lines examined, alterations in cell growth caused by lipophilic quaternary ions may involve KATP channels. We examined the effect of tetraphenylphosphonium (TPP), tetraphenylboron (TPB), rhodamine 123, dequalinium chloride (DECA) and the non-quaternary ion cisplatin on the proliferation of L1210 mouse leukaemia cells and rat smooth muscle cells in vitro. The KATP channel opener levcromakalim (LKM) and the KATP channel antagonist glibenclamide were also tested. 2. From growth-inhibition studies, the rank order of potency (based on pIC50 values) using L1210 leukaemia cells was: DECA (6.61) > cisplatin (6.09) = rhodamine 123 (6.01) > TPP (5.61) > TPB (4.25). Levcromakalim and glibenclamide were found to be inactive at the maximum concentrations used (100 mumol/L). A different rank order of potency was obtained in rat aortic smooth muscle cells: cisplatin (6.33) > DECA (5.67) > TPP (4.96) > rhodamine 123 (4.1). Tetraphenylboron (30 mumol/L), LKM (100 mumol/L) and glibenclamide (100 mumol/L) were found to be inactive. 3. When the negatively charged TPB (30 mumol/L) was combined with some of the active agents, the potency of the active agents was increased. Thus, in L1210 cells, rhodamine 123, DECA and TPP were all more potent at inhibiting cell growth in the presence of TPB. Tetraphenylboron had no effect on cisplatin in this cell line. In rat smooth muscle cells, TPB (30 mumol/L) potentiated the effect of rhodamine 123 but had no effect on the actions of cisplatin, DECA or TPP. 4. In functional studies, rhodamine 123 was a weak antagonist of the vasorelaxant responses to the KATP channel opener LKM in the porcine right circumflex artery in vitro. The pKB value obtained for rhodamine 123 at 100 mumol/L was 4.95. Dequalinium chloride was inactive. 5. We found no correlation between the actions of the compounds tested to antagonise KATP channels and their ability to inhibit cell proliferation. In addition, compounds known to regulate KATP channel activity failed to influence proliferative rates. These results suggest that KATP channels are not involved in the antiproliferative action of TPP and other quaternary ions in the two cell lines studied.

Cellular Responses in Mouse Leukemia L1210 Cells Made Resistant to Deoxyadenosine

Recent studies have implicated nucleotides in diverse and unexpected functions related to p53 levels, p53-dependent G0/G1cell cycle arrest, and the role of dATP in the activation of the caspase-induced apoptosis. Using deoxyadenosine-resistant L1210 cells (ED2 and Y8) that had ribonucleotide reductase that was not sensitive to inhibition by dATP and also exhibited other metabolic alterations, the properties of these cells with respect to the role(s) of nucleotides in these functions were explored. In the ED2 and Y8 cells that did not express p53 protein, the pools of UTP, CTP, ATP, and GTP were markedly decreased. The decreased cellular levels of UTP and CTP did not result in these cells being more sensitive to either PALA or acivicin. The ED2 and Y8 cells did not block in G0/G1in response to PALA treatment even though the basal cellular concentrations of UTP and CTP were reduced 50 to 80%. While it has been shown that dATP in combination with cytochrome c is involved in the apoptotic pathway, the concentration of exogenous deoxyadenosine required to induce apoptosis in the parental L1210 cells was far in excess of the concentration required to inhibit cell growth. Deoxyadenosine did not cause an increase in apoptosis in the deoxyadenosine-resistant Y8 cells. These data suggest that the new roles ascribed to nucleotides may be specific for the particular cell type under very specific conditions.

Oxidative DNA damage induced by visible light in mammalian cells: extent, inhibition by antioxidants and genotoxic effects

The extent of the indirect DNA damage generated in mammalian cells by visible light because of the presence of endogenous photosensitizers was studied by means of repair endonucleases. In immortalized human keratinocytes (HaCaT cells) exposed to low doses of natural sunlight, the yield of oxidative DNA base modifications sensitive to the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg protein) generated by this indirect mechanism was 10% of that of pyrimidine dimers (generated by direct DNA excitation). A similar yield of Fpg-sensitive modifications, which include 8-hydroxyguanine, was observed in primary keratinocytes. The relative yield of oxidative base modifications decreased at higher light doses, probably as a result of photodecomposition of the endogenous chromophore involved. For the three cell lines tested, viz. HaCaT cells, L1210 mouse leukemia cells and AS52 Chinese hamster cells, the yield of oxidative base modifications generated by a low dose of visible light appeared to be correlated with the basal concentrations of porphyrins in the cells. Induction of cellular porphyrin synthesis by pretreatment with 5-aminolaevulinic acid increased the light-induced oxidative damage in L1210 cells several-fold. In both induced and uninduced cells, the damage was inhibited by more than 50% in the presence of ascorbic acid (100 μM), while α-tocopherol and the iron chelator o-phenanthroline had no effect and β-carotene even increased the damage. Even high doses of visible light did not significantly increase the numbers of micronuclei in L1210 cells or of gpt mutations in AS52 cells. The negative outcome can be fully explained by the photobleaching of the endogenous photosensitizers, which prevents the generation of sufficiently high levels of oxidative DNA damage. Therefore, the mutagenic risk arising from the indirectly generated oxidative DNA modifications induced by sunlight may be underestimated when results obtained at high doses are extrapolated to low doses or low dose rates.