Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells

Because betel quid chewing has been linked to the development of oral cancer, pathobiological effects of an aqueous areca nut extract, four areca nut alkaloids (arecoline, guvacoline, guvacine, and arecaidine), and four nitrosated derivatives [N-nitrosoguvacoline, N-nitrosoguvacine, 3-(N-nitrosomethylamino)propionaldehyde and 3-(N-nitrosomethylamino)propionitrile] have been investigated using cultured human buccal epithelial cells. Areca nut extract in a dose-dependent manner decreases cell survival, vital dye accumulation, and membrane integrity, and it causes formation of both DNA single strand breaks and DNA protein cross-links. Depletion of cellular free low-molecular-weight thiols also occurs, albeit at quite toxic concentrations. Comparisons of the areca nut-related N-nitroso compounds and their precursor alkaloids, at concentrations up to 5 mM, indicate that 3-(N-nitrosomethylamino)propionaldehyde is the most potent on a molar basis to decrease both survival and thiol content and to cause significant formation of DNA single strand breaks. Arecoline, guvacoline, or N-nitrosoguvacoline decreases survival and cellular thiols, whereas arecaidine, guvacine, N-nitrosoguvacine, and 3-(N-nitrosomethylamino)propionitrile have only minor effects on these variables. Taken together, the present studies indicate that aqueous extract and, in particular, one N-nitroso compound related to areca nut, i.e., 3-(N-nitrosomethylamino)propionaldehyde, are highly cytotoxic and genotoxic to cultured human buccal epithelial cells, of potential importance in the induction of tumors in betel quid chewers.     

Analysis and pyrolysis of some N-nitrosamino acids in tobacco and tobacco smoke.

A new tobacco-specific nitrosamine, 4-(N-nitrosomethylamino)-4-(3-pyridyl)butyric acid (iso-NNAC), has been identified in tobacco, and its structure was confirmed by gas chromatography-mass spectrometry following enrichment of a tobacco extract. The levels of iso-NNAC ranged from 0.01 to 0.95 ppm. It does not induce DNA repair in primary rat hepatocytes and is inactive as a tumorigenic agent in strain A mice. In order to study the fate of nitrosamino acids during smoking, we spiked cigarettes with the following N-nitrosamino acids: iso-NNAC, 3-(nitrosomethylamino)propionic acid (NMPA), 4-(nitrosomethylamino)butyric acid (NMBA), N-nitrososarcosine (NSAR) and N-nitrosoproline (NPRO). NMPA and NMBA were partially transferred, unchanged, during smoking and partially formed the corresponding methyl esters, while pyrolysis of NSAR and NPRO resulted mainly in their decarboxylating products. This is the first time that the pyrosynthesis of methyl esters has been observed during smoking.     

Metabolism of benzo[a]pyrene by cultured rat and human buccal mucosa cells

Primary cultures of epithelial and fibroblast cells derivedfrom human oral mucosa were studied for the ability to activatea tobacco smoke carcinogen, benzo[a]pyrene (BP). The cells wereexposed to benzo[a]pyrene for 18 h. The cell-free medium wasextracted with ethylacetate/acetone, and high-pressure liquidchromatography analysis of this fraction revealed that BP tetrolsand diols were the major metabolites formed by both epithelialand fibroblast cells. However, the epithelial cells had a muchhigher rate of biotransformation of BP as measured by bindingto cellular DNA. The mean binding level to human buccal mucosalDNA was among the highest observed in stratified human epithelia.The major BP - DNA adduct was formed by the reaction of the‘bay-region’ BP diolepoxide with the exocyclic 2-aminogroup in guanine. In contrast to human cells, BP phenols andBP 9, 10-diol were the major metabolites produced by primaryepithelial and fibroblast cells derived from rat buccal mucosa.The DNA binding levels of BP in the two rat cell types wereidentical, and the binding level was several-fold lower thanin the human epithelial cells. When an established rat tongueepithelial cell line (RTE 2) was treated with polycyclic aromatichydrocarbons - BP and 7,12-dimethylbenz[a]-anthracene - a slighttoxic effect was observed. Our results indicate that primarycultures of oral mucosa are able to metabolize BP into its ultimatecarcinogenic form at a rate similar to or higher than otherpotential target tissues for BP-induced carcinogenesis.     

Expression and DNA methylation changes in human breast epithelial cells after bisphenol A exposure

It has been suggested that xenoestrogens, a group of agents termed endocrine disruptors, may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. We previously demonstrated that the xenoestrogen, bisphenol A (BPA), was able to induce the transformation in vitro of human breast epithelial cells. The normal-like human breast epithelial cell line, MCF-10F, formed tubules in collagen (3-D cultures), although after treatment with BPA (10-5 M and 10-6 M BPA) the cells produced less tubules (73% and 80%, respectively) and some spherical masses (27% and 20%, respectively). In the present study, expression and DNA methylation analyses were performed in these cells after exposure to BPA. These cells showed an increased expression of BRCA1, BRCA2, BARD1, CtIP, RAD51 and BRCC3, all of which are genes involved in DNA repair, as well as the downregulation of PDCD5 and BCL2L11 (BIM), both of which are involved in apoptosis. Furthermore, DNA methylation analysis showed that the BPA exposure induced the hypermethylation of BCL2L11, PARD6G, FOXP1 and SFRS11, as well as the hypomethylation of NUP98 and CtIP (RBBP8). Our results indicate that normal human breast epithelial cells exposed to BPA have increased expressions of genes involved in DNA repair in order to overcome the DNA damage induced by this chemical. These results suggest that the breast tissue of women with BRCA1 or BRCA2 mutations could be more susceptible to the effects of BPA.     

Acute inhibition of DNA synthesis in hamster buccal pouch epithelium exposed to indirect acting carcinogens

Inhibition of DNA synthesis was observed and quantitated in hamster buccal pouch epithelium exposed in vivo and in vitro to indirect acting carcinogens. Topical application of a 0.5% solution of the potent hamster buccal pouch carcinogen 7,12-dimethylbenz[a]-anthracene (DMBA) acutely inhibited epithelial DNA synthesis by 40-65%, as indicated by a decrease in [3H]thymidine incorporation over a period of 24 h. When applied twice at a concentration of 2%, N-methyl-N-benzylnitrosamine (MBN), another potent buccal pouch carcinogen, inhibited epithelial DNA synthesis by 76% within a period of 4 h. A similar acute inhibitory effect on DNA synthesis was observed when explants of buccal pouch mucosa, exhibiting continuous cell replication, were exposed in vitro in the presence of MBN or DMBA for periods up to 12 and 24 h, respectively. The inhibitory effect of DMBA was greater than that of other polycyclic aromatic hydrocarbons of lesser carcinogenic potency in this tissue. This study demonstrates that the metabolic activation of indirect acting carcinogens leading to acute cytotoxicity and inhibition of DNA synthesis occurs rapidly in hamster buccal pouch mucosa exposed to these agents in vitro as well as in vivo. The experimental imposition of an acute inhibitory pressure, applied as demonstrated in this report, may enable the detection of precancerous cells which have acquired the property of resistance to this cytotoxic effect in the course of carcinogenesis. In principle, the in vitro approach, coupled with autoradiography, may be useful in identifying microscopic foci of resistant preneoplastic cells in samples of human oral mucosa. 24R01 34160     

Quinoxaline 1,4-dioxides as anticancer and hypoxia-selective drugs

Hypoxic cells which are found in solid tumors are resistant to anticancer drugs and radiation therapy. Thus, for effective anticancer chemotherapy, it is important to identify drugs with selective toxicity towards hypoxic cells. Quinoxaline 1,4-dioxides (QdNOs) are heterocyclic aromatic N-oxides that have been found to possess potent antibacterial activities (inhibit microbial DNA synthesis) especially under anaerobic conditions; thus they are under evaluation as bioreductive drugs for the treatment of solid tumors (1). We investigated the ability of four differently substituted QdNOs to inhibit cell growth and induce cell cycle changes in two human tumorigenic epithelial cell lines under oxic conditions. We also evaluated the toxicity of these drugs to cancer cells cultured under hypoxic conditions. Two epithelial cell lines (the T-84 human colon cancer-derived cell line, and the SP-1 keratinocyte cell line) were treated with various doses of the QdNOs and harvested at different times after treatment. Proliferation and cell cycle results showed a structure-function relationship in the activity of the various QdNO compounds with the 2-benzoyl-3-phenyl-6,7-dichloro-derivative of QdNO (DCBPQ) being the most potent cytotoxin and hypoxia-selective drug. The 2-benzoyl-3-phenyl (BPQ) and the 2-acyl-3-methyl-derivative of QdNO (AMQ) were less cytotoxic but arrested almost 50% of the cells in the G2M phase of the cell cycle at doses of 30 and 120 microM, respectively. The tetramethylene derivative of QdNO (TMQ) did not affect the growth and cycling of cells cultured in air and was the least potent cytotoxin to hypoxic cells. Our results indicate that the QdNOs are hypoxia-cytotoxic drugs whose activity varies according to the substituents on the quinoxaline 1,4-dioxide heterocycle. Because of their selective toxicity to hypoxic cells (cells found in human tumors), these drugs may provide useful therapeutic agents against solid tumors.     

Characterization of activation and deactivation pathways of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) in rat hepatocytes

We have characterized the metabolism of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) in cultured rat hepatocytes and have established the relationship between various metabolic pathways and single-strand breaks (SSB) in DNA. Metabolism of [5-3H]-NNK by carbonyl reduction, alpha-carbon hydroxylation and pyridine N-oxidation was linear from 0.5 to 6 h with 0.25-2 x 10(6) hepatocytes. Using an alkaline elution assay, we observed that NNK induces SSB in DNA in a dose- and time-dependent manner. SSB induced by NNK were rejoined partially within 2 h and totally by 12 h after exposure. NNK-N-oxide produced a smaller number of SSB than NNK, suggesting that pyridine N-oxidation of NNK is a deactivation pathway. Carbonyl reduction of NNK led to 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butan-1-ol (NNAl). Reaction of NNK with methyl magnesium iodide gave 4-(N-nitrosomethylamino)-1-(methyl)-1-(3- pyridyl)butanol-1-ol (1-MeNNAl) 82% yield. NNAl, but not 1-MeNNA1, can be reoxidized to NNK. Doses of 5 mM NNAl and 1-MeNNAl both induced SSB, indicating that NNAl does not require reconversion to NNK to be activated to DNA damaging intermediates. alpha-Methylene hydroxylation resulted in the formation of 4-oxo-4-(3- pyridyl)butanal. At equimolar concentration (5 mM), the aldehyde was more damaging than NNK to hepatocyte DNA. The results of this study demonstrate that NNK is activated by rat hepatocytes and that metabolites formed by alpha-carbon hydroxylation induce SSB.     

Relationship between cellular levels of beta-carotene and sensitivity to genotoxic agents

The usefulness of an in vitro test system to predict the inhibitory effect of beta-carotene on the genotoxic activity of carcinogens/mutagens was explored. To facilitate the comparison of data obtained from cultured cells (CHO) and from exfoliated human cells, endpoints were used which can be quantitated in both cell systems: the frequency of micronuclei for estimating the effect of genotoxic agents, and cellular levels of beta-carotene as a protective agent. In CHO cells, beta-carotene inhibited the clastogenic and micronucleus-forming effect of methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4NQO), but had no protective action against gallic acid, tannic acid, and aqueous extract of areca nut or H2O2. The extent of inhibition depended on the ratio of beta-carotene to MMS. Doses of beta-carotene which exerted a protective effect in vitro ranged from approximately 2 to 5 ng per 10(6) CHO cells. Comparable levels of beta-carotene were previously found to reduce the frequency of micronucleated exfoliated cells from the buccal mucosa of tobacco and areca-nut chewers (Stich et al., 1984b).     

Antitumor activity and mechanism of action of the novel marine natural products mycalamide-A and -B and onnamide

Three novel heterocyclic compounds, mycalamide-A and -B and onnamide, were isolated from Mycale sp. and Theonella sp. sponges collected in New Zealand and Okinawan waters. Each exhibited potent in vitro toxicity and in vivo efficacy against murine and human tumor cells. Concentrations of each that inhibited replication of cultured murine lymphoma P388 cells by 50% were 5 nM or less. Mycalamide-A and -B were also potent inhibitors of HL-60, HT-29, and A549 human tumor cell replication (50% inhibitory concentration less than 5 nM), while values for onnamide were greater (50% inhibitory concentrations between 25 and 200 nM). Mycalamide-A (10 micrograms/kg) and -B (2.5 micrograms/kg) were moderately active against P388 leukemia (increase in life span, approximately 50%), while onnamide was inactive (40 micrograms/kg; increase in life span, 15%). Mycalamide-A was also active against B16 melanoma, Lewis lung carcinoma, M5076 ovarian sarcoma, colon 26 carcinoma, and the human MX-1, CX-1, and Burkitt's lymphoma tumor xenografts. Mechanism of action studies indicate that the three agents inhibited protein synthesis. For example, after 1-h exposures to 20 nM mycalamide-A and -B, the rates of [3H]leucine incorporation into acid-precipitable material of cultured P388 cells were inhibited 54 and 99%, while the effects on incorporation of [3H]uridine and [3H]thymidine were less. The relative effects of 20 to 2000 nM mycalamide-A on protein, RNA, and DNA synthesis were consistent with those observed during exposure of P388 cells to 1 microM emetine, a known inhibitor of protein synthesis. Also, the three agents inhibited translation of RNA into protein in a cell-free lysate of rabbit reticulocytes. Although mycalamide-A disrupted DNA metabolism, the agent apparently did not intercalate into DNA, and a mixture of four deoxynucleosides (250 microM each) did not decrease the antiproliferative effects of the agent. Collectively, these data indicate that this class of compounds represents novel antitumor agents which should be further evaluated to define their potential.     

Localization of DNA adducts formed in the nasal cavity of the rat by the tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK)

The tissue localization of the DNA adducts O6- and 7-methylguanine induced in the nasal cavity by the nicotine-derived carcinogen 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK, 30 mg/kg intraperitoneally) has been investigated immunocytochemically in male Sprague-Dawley rats. Adduct-specific nuclear staining, indicative of the metabolic activation of NNK to a methylating compound, was observed in both respiratory and olfactory mucosa. In the respiratory epithelium, strong staining was generally observed in areas devoid of goblet cells. Less intense staining was observed both in the serous gland cells and their efferent ducts in the respiratory submucosa, whereas the mucous gland cells were unstained. In the olfactory mucosa, the sustentacular and basal cells of the olfactory epithelium were moderately stained; staining varied substantially from site to site. No DNA adduct was detected in the olfactory cells. Strong nuclear staining, similar to that in the respiratory mucosa, was observed in the cells of the Bowman glands of the olfactory submucosa. A similar distribution of methylated DNA bases in nasal tissues has been observed in rats after exposure to other N-nitrosamines and in Syrian hamsters after exposure to NNK. This finding may indicate that in man the same cell types undergo DNA adduct formation after exposure to NNK and other N-nitrosamines.     

Pathobiological effects of acrolein in cultured human bronchial epithelial cells

The ability of the highly reactive aldehyde acrolein to affect growth, membrane integrity, differentiation, and thiol status and to cause DNA damage has been studied at serum- and thiol-free conditions using cultured human bronchial epithelial cells. Acrolein markedly decreases colony survival at 3 microM whereas about 10-fold higher concentrations are required to increase membrane permeability, measured as uptake of trypan blue dye. Acrolein at micromolar concentrations also causes epithelial cells to undergo squamous differentiation as indicated by decreased clonal growth rate, dose-dependent increased formation of cross-linked envelopes, and increased cell planar surface area. Acrolein causes a marked and dose-dependent cellular depletion of total and specific free low-molecular-weight thiols as well as protein thiols. Exposure to acrolein did not cause oxidation of glutathione indicating that thiol depletion occurred by direct conjugation of reduced glutathione to acrolein without concomitant generation of active oxygen species. Furthermore, acrolein is genotoxic and causes both DNA single strand breaks and DNA protein cross-links in human bronchial epithelial cells. The results indicate that acrolein causes several cytopathic effects that relate to multistage carcinogenesis in the human bronchial epithelium.     

Investigations on the molecular dosimetry of tobacco-specific N-nitrosamines

Approaches for assessing molecular dosimetry of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) in humans by measurement of haemoglobin or DNA adducts are discussed. NNK and NNN form haemoglobin adducts in Fischer 344 rats. Acid or base hydrolysis of the globin gives 4-hydroxy-1-(3-pyridyl)-1-butanone, which can be detected in rat blood up to six weeks after injection of NNK; it may be a useful marker for assessing uptake and metabolic activation of NNK and NNN in tobacco consumers. NNK and its major metabolite, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAI), methylated DNA of rat liver, lung and nasal mucosa to similar extents. NNAI is formed in human tissues from NNK, but immunoassays for O6-methyldeoxyguanosine (O6-medGuo) in exfoliated oral cells from snuff-dippers have been negative. NNK is also expected to form pyridyloxobutyl adducts in DNA; 32P-postlabelling assays for these adducts are being developed and appear to hold promise for detecting NNK- or NNN-DNA adducts in vivo.     

Pathobiological effects of aldehydes in cultured human bronchial cells

Effects of the tobacco smoke-related aldehydes, i.e., acetaldehyde, formaldehyde and acrolein, have been investigated in cultured human bronchial epithelial cells and fibroblasts. As determined from loss of colony-forming efficiency of epithelial cells, acrolein is 200- and 5000-fold more toxic than formaldehyde and acetaldehyde, respectively. The aldehydes differ markedly in their potencies to induce terminal differentiation, as indicated by cessation of growth and enhanced formation of cross-linked envelopes. The cellular content of glutathione is markedly decreased by acrolein, whereas formaldehyde and acetaldehyde only slightly decrease glutathione levels. All three aldehydes produce DNA damage, as indicated by the formation of DNA single-strand breaks and DNA protein cross-links. Both formaldehyde and acrolein are weakly mutagenic in fibroblasts. In-vitro assays of O6-methylguanine-DNA methyltransferase (MMT) activity indicate that it is markedly inhibited by acrolein, and to a lesser extent by formaldehyde. However, formaldehyde significantly inhibits removal of O6-methylguanine (O6-meG) in N-methyl-N-nitrosourea (MNU)-exposed cells. Thus, the many biological effects induced by aldehydes include: inhibition of proliferation, enhanced cellular differentiation, thiol depletion, DNA damage, mutation and inhibition of DNA repair in human cells. Furthermore, we speculate that exogenous or metabolically generated aldehydes may increase the genotoxicity of N-nitroso compounds by the dual action of causing DNA damage and inhibiting the repair of promutagenic O6-meG DNA lesions in human cells.     

Monitoring of sister chromatid exchange and micronuclei as biological endpoints

Cytologically visible damage in human chromosomes can be detected as structural chromosomal aberrations, numerical changes in genome, sister chromatid exchanges (SCE) or as micronucleated cells. The importance of in-vivo cytogenetic damage that is induced in human cells is that it indicates that similar alterations may have occurred in other tissues, either in somatic or in germinal cells. SCEs represent symmetrical exchanges between sister chromatids; generally, they do not result in alteration of the chromosome morphology or the genetic information. Although the detection method is highly sensitive as an in-vitro screening test, in monitoring studies, it seems to be restricted to cases where the exposing agents are strong alkylating compounds (e.g., ethylene oxide, cytostatic drugs) or to some multi-exposure conditions (e.g., cigarette smoking, laboratory work, rubber industries). Micronuclei arise from acentric chromosome fragments or lagging whole chromosomes. They have been detected in a variety of dividing human cells, including exfoliated epithelial cells, bone-marrow cells and lymphocytes. Positive responses of induction of cells with micronuclei have been obtained in studies with cells of the buccal mucosa (chewers of betel or tobacco) or cultured lymphocytes of some groups occupationally exposed to agents like styrene or ethylene oxide.     

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water- based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well- established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.      

Effects of tumor promoters and cocarcinogens on growth and differentiation of cultured human esophageal epithelial cells

The acute effects of 12-O-tetradecanoylphorbol-13-acetate [(TPA) CAS: 56937-68-9], T-2 toxin (CAS: 21259-20-1), capsaicin (CAS: 404-86-4), cigarette smoke condensate (CSC), and ethanol (CAS: 3807-77-0) were examined in secondary cultured human esophageal epithelial cells in serum-free LHC-8 medium. Effects were evaluated by morphology and measurement of clonal growth rate (population doublings per day), cross-linked envelope (CLE) formation, and the enzymatic activities of ornithine decarboxylase (ODC) and plasminogen activator (PA). All compounds tested were inhibitory to clonal growth; concentrations causing 50% growth inhibition were estimated as 10 nM TPA, 6 nM T-2 toxin, 40 microM capsaicin, 8 micrograms CSC/ml, 540 mM ethanol, and 0.8 microgram CSC/ml with 220 mM ethanol. None of the compounds tested induced CLE formation, although calcium ionophore (A23187) could induce CLE in at least 60% of the cells. TPA (10 and 100 nM) decreased the ODC activity of cells, and capsaicin (100 microM) induced ODC by 220%. TPA (1-100 nM) and capsaicin (100 microM) also induced PA activity. Slight increases in ODC activity by CSC (10 micrograms/ml), CSC (1 microgram/ml) with ethanol, and T-2 toxin (1 nM) were observed, but PA activity was not affected by these compounds. The results indicated that the response of human esophageal epithelial cells to TPA is both similar to and different from that reported for human epidermal and bronchial cells in vitro. Enhancement of PA activity and decrease in ODC by TPA are found in all three human epithelial cell types. However, these changes are not associated in esophageal cells with increased CLE formation as reported in studies with the use of bronchial and epidermal epithelial cells. The results from these acute studies provide the basis for designing in vitro carcinogenesis investigations using these agents.     

Monitoring of interaction products of cis-diamminedichloroplatinum(II) and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) with DNA in cells from platinum-treated cancer patients

The formation and stability of interaction products between the anti-cancer drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA were studied in buccal epithelial and urinary cells from ten cancer patients who received cis-DDP-based therapy. Buccal cells were collected 1 h before and 1-2 h after i.v. infusions with cis-DDP. The interaction products were visualized in an immunocytochemical peroxidase assay, using an antiserum against cis-DDP-modified calf thymus DNA. The nuclear staining density was measured by microdensitometry. Nuclear staining densities in buccal cells after infusions of greater than or equal to 20 mg/m2 cis-DDP were always higher than pretreatment values. Repeated sampling from individual patients treated for 2-5 consecutive days with daily doses of 20-70 mg/m2 cis-DDP indicated that cis-DDP-DNA binding in buccal cells increased in proportion to the cumulative total dose of cis-DDP. The variation in dose-density response between patients was 17%. Apparent adduct loss in buccal cells from four patients, as measured 8-17 days after the last infusion, amounted to 67-86%. Platinum-induced DNA modifications could also be detected in buccal cells from two cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)-treated patients. In vitro experiments with human buccal cells and lymphocytes indicated linear relationships between DNA modification and either cis-DDP concentration or incubation time. Nuclear staining densities in pretreatment buccal cells from ten cancer patients treated in vitro with 33 microM cis-DDP for 1 h revealed that interpatient variation in in vitro DNA modification by cis-DDP was low. No quantitative correlation was found between in situ and in vitro DNA modification.     

DNA damage induced by a new 2-chloroethyl nitrosourea on malignant melanoma cells

Different biological aspects of a novel 2-chloroethyl nitrosourea derived from cysteamine, N'-(2-chloroethyl)-N-[2-(methylsulfinyl)ethyl]-N'- nitrosourea (CMSOEN2), were studied. Drug-induced cytotoxic effects, uptake kinetics, DNA damage, and O6-alkylguanine-DNA alkyltransferase activity were determined in 3 melanoma cell lines: the murine B16 and 2 human metastatic-derived cell lines (M4 Beu and M3 Dau). We found that radioactivity uptake and incorporation in acido-precipitable material was inversely proportional to cell drug viability. The highly CMSOEN2-sensitive B16 line showed the lowest total radioactivity uptake. In fact, among the melanoma cell parameters studied, 3 of them were well correlated: (a) cytotoxicity as reflected by the colony-forming assay; (b) DNA cross-link frequency estimated by the alkaline elution technique; and (c) O6-alkylguanine-DNA alkyltransferase activity (Mer phenotype), defined as the ability of cell extracts to remove O6-methylguanine from N-methyl-N-nitrosourea-alkylated DNA. The 2 human cell lines (M4 Beu and M3 Dau), the most resistant to the cytostatic drug effects, showed little or no ability to form DNA lethal cross-links. These results correspond to the higher O6-alkylguanine-DNA alkyltransferase activity found in human-derived cell lines compared with that present in murine B16 cell lines. This study confirms that the cell content in this repair DNA protein is certainly one of the important factors implicated in the variability of response to 2-chloroethyl nitrosourea treatment observed in a number of established malignant cell lines. It has been shown that pretreatment of derived cell lines with methylating agents (N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine) or O6-methylguanine used as a free base, increased cytotoxic effects of this class of anticancer agents, likely by saturating receptor sites (sulfhydryl groups) of this specific DNA repair enzyme. Nevertheless, in preliminary Phase I and II clinical trials, 2 patients who had been treated with multiple chemotherapies including alkylating agents [1-(2-chloroethyl)-3- cyclohexyl-1-nitrosourea, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, platinum derivatives], presented complete or partial remission after CMSOEN2 treatment. Our results raise the question of the exact relation between the Mer phenotype determined in derived murine or human cultured cells and that directly observed on surgically excised tumors in cancer patients. The original Mer phenotype could be modified by cell culture conditions since it has been shown that O6-alkylguanine-DNA alkyltransferase activity is widely distributed between normal and tumoral tissues without any real difference.     

Metabolic activation of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to DNA binding species in human mammary epithelial cells.

When incubated in suspension with the heterocyclic aromatic amine food mutagens 2-amino-3-methylimidazo [4,5-f]-quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), human mammary epithelial cell aggregates were found, by 32P-postlabelling analysis, to yield DNA that contained adducts. Analysis by HPLC of the 32P-labelled digests of mammary cell DNA indicated that in each case a major adduct peak corresponded to that produced in DNA in vitro by activated derivatives of the two compounds. The patterns of adducts obtained when DNA digests were separated by TLC on polyethyleneimine-cellulose plates were found to resemble those previously shown to be present in DNA of tissues of mice fed IQ or MeIQ. These results demonstrate the ability of human mammary epithelial cells to activate carcinogenic heterocyclic compounds known to be present in the human diet to DNA binding derivatives.     

Approaches to the development of assays for interaction of tobacco-specific nitrosamines with haemoglobin and DNA

The tobacco-specific, nicotine-derived nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are among the most important carcinogens in tobacco and tobacco smoke. Treatment of Fischer 344 rats with these carcinogens resulted in alkylation of haemoglobin and DNA by the 4-(3-pyridyl)-4-oxobutyl group formed during their metabolism. This alkyl group can be detached from globin or DNA under mild hydrolytic conditions as 4-hydroxy-1-(3-pyridyl)-1-butanone, which appears to be a potentially useful dosimeter for human exposure to, and activation of, tobacco-specific nitrosamines.