Human melanoma cells sensitive to deoxyadenosine and deoxyinosine

In an in vitro study conducted without the use of adenosine/deoxyadenosine deaminase inhibitors, two human melanoma cell lines, MM96L and MM127, were found to be highly sensitive to killing by continuous treatment with deoxyadenosine (dAdo) (D37 47 microM and 68 microM respectively) compared with fibroblasts (D37 440 microM), Hela cells (D37 1.1 mM) and other melanoma cell lines (D37 0.8 to 2.5 mM). Cross-sensitivity was found to deoxyinosine (dIno) and in part to adenosine but not to related metabolites such as inosine or hypoxanthine. Hypersensitivity to dAdo was associated with deficiency in cell membrane 5'-deoxynucleotidase but not in deaminase activity. dAdo toxicity could be prevented in MM96L by addition of the other three deoxynucleosides together but not by removing dAdo after a brief (2 hr) treatment. Resistant melanoma cells, however, required more than 24 hr dAdo treatment to produce toxicity. DNA synthesis in MM96L cells was reversibly inhibited, and cells tended to accumulate in G1/S. No DNA strand breaks were detected. These results showed that in contrast to the resistant cell line, asynchronous MM96L cells are highly sensitivity to brief treatment, toxicity resulting from an effect associated with inhibition of DNA synthesis. dAdo and dIno, either combined with a deaminase inhibitor or as deaminase-resistant derivatives, may have a favourable therapeutic index for some melanomas in vivo.     

Inhibitory effect of rhodamine B on the proliferation of human lip fibroblasts in culture

The effect of the cosmetic dye rhodamine B on the proliferation of human lip fibroblasts (KD cells) was investigated in a culture system. Rhodamine B at 25 micrograms/ml and above significantly decreased the number of the cells after a 72 h culture. A time course study revealed that 50 micrograms/ml of rhodamine B-induced decrease in the cell number occurred after 48 h and longer, suggesting that the dye inhibited cell proliferation without a decrease in cell attachment. The detachment of [3H]thymidine-labeled cells from the monolayer was unaffected by rhodamine B at 100 micrograms/ml and below. The incorporation of [3H]thymidine and [14C]leucine into the acid-insoluble fraction of the cell layer was significantly inhibited by 50 micrograms/ml rhodamine B treatment. Histologically, the damage of KD cells was not marked, however, a degenerative change of nuclei and an irregular shape of the cells as well as a decrease in the cell number were caused by 50 micrograms/ml rhodamine B. Rhodamine 6G caused a severe damage of the cells, and rhodamine B significantly decreased the cell number; rhodamine 123 had no significant effect; rhodamine 116 significantly increased the cell number. Furthermore, rhodamine B decreased the number of both vascular endothelial cells from bovine aorta and vascular smooth muscle cells from murine aorta after a 72 h culture. It is concluded that rhodamine B inhibits the proliferation of human lip fibroblasts. This rhodamine B effect may be a warning sign for the dye toxicity.     

Effects of methimazole on human lymphocyte proliferation and natural killer cell activity

Methimazole (MM), an antithyroid drug, was examined for its ability to modulate immune functions. The increase in [3H]thymidine incorporation in plant mitogen-stimulated lymphocytes and augmentation of NK cell activity by MM were used as a measure of the immunomodulatory activity of MM. The results demonstrated that lymphocytes from 5 different donors had a mean increase of 632% of [3H]thymidine incorporation in the presence of MM at a concentration of 114.2 micrograms/ml (P less than 0.005). Lymphocyte response to PHA, Con A or PWM was potentiated by 27 to 482% in the presence of MM. The MM mediated increase in lymphocyte proliferation was more obvious in cases where the mitogenic stimulation was low. Lymphocyte cytotoxicity to both K562 cells and malignant B cells was also augmented when lymphocytes were cultured in the presence of MM. A maximal effect was observed when lymphocytes were treated with a concentration of 20 micrograms/ml of MM for 12-16 hr. Maximum augmentation was usually exerted when the NK cell activity of cultured lymphocytes was low. This was particularly seen with lymphocyte from cultured B cell leukemia (3163).     

Effects of complex of 3,6-di-(dimethylamino)-dibenzopyriodonium with praseodymium dicitrate on the syntheses of DNA, RNA, protein, nucleoprotein and ATP of leukemia L 7712 cells in mice

The incorporations of [3H] thymidine, [3H]uridine and [3H]leucine into DNA, RNA and protein synthesis in leukemia 7712 cells were inhibited by the complex of 3,6-di-(dimethylamino)-dibenzopyriodonium with praseodymium (Pr, rare earth element) dicitrite 34 micrograms/ml for 3-24 h. The degree of inhibition increased in proportion to the incubation time. After being treated with [C17H20N2I]3[Pr(C6H5O7)2] 34 micrograms/ml for 3, 6, 12 and 24 h, the incorporation of [32P]Na2HPO4 into the nucleoprotein of leukemia 7712 cells was inhibited by 49, 57, 65 and 85%, while those into ATP were inhibited by 43, 59, 65 and 83%, respectively. The ID50 of [C17H20N2I]3[Pr(C6H5O7)2] on DNA synthesis in leukemia 7712 cells at 24 h was 22 micrograms/ml. After the complex was removed from the medium entirely, the rate of DNA synthesis decreased with time over 3-12 h. This result indicated that the inhibition mechanism was likely due to damage to the DNA template.     

Rhodamine B inhibition of glycosaminoglycan production by cultured human lip fibroblasts

Rhodamine B (Rh B) is a dye which is used in cosmetics such as lipsticks. We investigated the effect of the dye on the metabolism of [3H]glucosamine-labeled glycosaminoglycans ([3H]-GAG) in confluent cultures of human lip fibroblast KD cells. It was found that Rh B at 10 micrograms/ml and above significantly decreased the accumulation of [3H]-GAG in both the cell layer and the medium after a 24-hr culture. This Rh B (50 micrograms/ml) effect was recognized in the cell layer and the medium after 8 and 24 hr, respectively, and longer. The Rh B at the 25 micrograms/ml-induced decrease in the [3H]-GAG accumulation was reversible in both the cell layer and the medium. On the other hand, the release of [3H]-GAG from the cell layer was unaffected by Rh B. A characterization of [3H]-GAG revealed that all components of the GAG including hyaluronic acid, heparan sulfate, chondroitin 4- and/or 6-sulfate, and dermatan sulfate were significantly decreased by 50 micrograms/ml Rh B in both the cell layer and the medium. Rh B significantly decreased the accumulation of [3H]-GAG in the presence of either 10 microM cycloheximide or 1 mM p-nitrophenyl-beta-D-xyloside, suggesting that Rh B inhibited the sugar chain formation rather than core protein synthesis. Although the number of confluent KD cells was significantly decreased by Rh B at 10 micrograms/ml and above, this Rh B effect was much weaker than that on the [3H]-GAG accumulation. The release of lactate dehydrogenase from the cell layer was significantly increased by 100 micrograms/ml Rh B but not by the dye at 75 micrograms/ml and below. From these results, it was suggested that Rh B decreases the GAG content of human lip fibroblasts through a functional suppression of polysaccharide chain formation in the process of the GAG production.     

Effects of some mycotoxins on mitogen-induced blastogenesis and SCE frequency in human lymphocytes

The effects of T-2 toxin, diacetoxyscirpenol, ochratoxin A and zearalenone on DNA synthesis in phytohaemagglutinin-stimulated human peripheral blood lymphocytes were studied by assaying the incorporation of [3H]thymidine. Total inhibition was obtained with 8 ng T-2 toxin/ml, 8 ng diacetoxyscirpenol/ml or 30 micrograms zearalenone/ml, and with 20 micrograms ochratoxin A/ml inhibition was almost complete; 50% inhibition was produced by 1.5 ng T-2 toxin/ml, 2.7 ng diacetoxyscirpenol/ml, 14 micrograms zearalenone/ml or 14 micrograms ochratoxin A/ml. The toxicity of the trichothecenes to the lymphocytes was slightly reduced when rat liver cells were present whereas the toxicity of ochratoxin A and zearalenone was unaltered. Low concentrations of trichothecenes did not produce any inhibition of DNA synthesis when the cultures were washed and placed in fresh media containing only phytohaemagglutinin 4 hr after the addition of the test compounds. Sister chromatid exchange frequency was not elevated by T-2 toxin, diacetoxyscirpenol or ochratoxin A. Zearalenone had a weak enhancing effect on the frequency of sister chromatid exchanges.     

Effects of actinomycin D analogs on nucleolar phosphoprotein B23 (37,000 daltons/pI 5.1)

Localization of protein B23 in HeLa cells after treatment with actinomycin D and its analogs was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with actinomycin D (250 ng/ml) for 2 hr, a uniform nucleoplasmic fluorescence was observed. Similar results were obtained with the actinomycin analogs, actinomycin Z5 and actinomycin K2T. Only after a much longer incubation (24 hr) with actinomycin 4-4'-gly was nucleoplasmic fluorescence observed. Actinomycin D, actinomycin Z5, and actinomycin K2T inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with IC50 values of 9.5 +/- 3.2, 59.1 +/- 19.6 and 1423.3 +/- 212.2 ng/ml respectively. No inhibition of [3H]uridine incorporation was observed using actinomycin 4-4'-gly (2000 ng/ml, 2-hr incubation). The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was actinomycin D greater than actinomycin Z5 greater than actinomycin K2T greater than actinomycin 4-4'-gly, which correlated with the order of their IC50 values for inhibition of [3H]uridine incorporation. Studies of the effects of actinomycin D and its analogs on RNA synthesis and localization of protein B23 indicated that there is a direct relationship between the B23 "translocation" from nucleolus to nucleoplasm and the inhibition of RNA synthesis. At 45-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. At 75-85% inhibition, only a uniform nucleoplasmic fluorescence was observed.     

Antiviral activity and its mechanism of guanine 7-N-oxide on DNA and RNA viruses derived from salmonid

Guanine 7-N-oxide produced by Streptomyces sp. was found to inhibit in vitro the replication of herpes virus (Oncorhynchus masou virus, OMV), rhabdo virus (infectious hematopoietic necrosis virus, IHNV) and a bi-segmented double-strand virus (infectious pancreatic necrosis virus, IPNV) derived from salmonids with IC50 values of about 10 micrograms/ml, 20 micrograms/ml and 32 micrograms/ml, respectively. The agent was not toxic for the host cells (chinook salmon embryo, CHSE-214) at the IC50 concentrations. Labeling of IHNV viral RNA and host cellular DNA and RNA with [3H]uridine and [3H]thymidine during drug treatment showed that guanine 7-N-oxide did not reduce the incorporation of these precusors into RNA and DNA. The anti-IHNV activity of guanine 7-N-oxide was enhanced synergistically by neplanocin A, an inhibitor of RNA methylation. The mechanism of action of guanine 7-N-oxide is discussed, in regard to maturation of viral messenger RNA including capping.     

Terpentecin, an inhibitor of DNA synthesis

Terpentecin at a concentration of 0.78 microgram/ml decreased the number of viable cells of Escherichia coli NIHJ to less than one thousandth the starting number in an hour when added to an exponentially growing culture in a nutrient broth. During this time, the turbidity of the cell suspension kept increasing as fast as the control. Microscopic inspection of the cells exposed to terpentecin under these conditions revealed that the cells were elongated. Terpentecin at a concentration of 6.25 micrograms/ml inhibited incorporation of [14C]thymidine into the acid-insoluble material of cells of E. coli NIHJ by 70% in 30 minutes in contrast to little or no inhibition of the incorporation of [14C]uridine or [14C]leucine. Under similar conditions, terpentecin did not inhibit either membrane transport (uptake) of [14C]thymidine into the cells or the metabolic conversion of the precursor into various cellular acid-soluble components. Terpentecin at a higher concentration (70 micrograms/ml) inhibited by 40% in 30 minutes the incorporation of [methyl-3H]thymidine triphosphate into the DNA fraction of toluene-treated cells of E. coli JE6296 (pol A-). Terpentecin showed stronger antibacterial activities against Bacillus subtilis M45T (rec-) and E. coli BE1121 (rec A-) than against their corresponding wild type strains. However, terpentecin showed no mutagenicity by the Ames test with Salmonella typhimurium strains TA100, TA98, TA92, TA1538, TA1537 and TA1535, and with E. coli WP2 (uvr A). Terpentecin at a lower concentration (0.07 micrograms/ml) inhibited growth in vitro of mouse leukemia L1210 cells by 50%. With the mammalian cells again the incorporation of [14C]thymidine into the acid-insoluble cell material was inhibited more strongly than incorporation of [14C]uridine and [14C]leucine. There was no sign of mutagenicity by the micronucleus test using mice.     

Metabolism of 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamide and related five-membered heterocycles to 5'-triphosphates in human blood and L5178Y cells.

The facile metabolism of 5-amino-1-β-d-ribofuranosylimidazole-4-carboxamide (AICA ribonucleoside) and several of its structural analogs [5-fluoro-1-β-d-ribofuranosylimidazole-4-carboxamide (FICA ribonucleoside), pyrazofurin and ribavirin] to their corresponding 5′-triphosphates in human blood cells has been demonstrated in vitro. Evidence is presented that both the β- and α-anomeric forms of pyrazofurin nucleotides were present in the extracts of pyrazofurin-treated blood. Determination of the extent of incorporation of radioactivity from [U-¹⁴C]d-glucose into analog ribonucleoside 5′-triphosphates formed in human blood cells indicated that AICA ribonucleoside and ribavirin were metabolized to their 5′-monophosphates mainly (> 90 per cent) via a nucleoside kinase; however, FICA ribonucleoside appeared to be metabolized to its 5′-monophosphate both via a nucleoside kinase (ca. 67 per cent) and via phosphorolytic cleavage followed by a phosphoribosyltransferase-mediated reaction (ca. 33 per cent). The aglycones of AICA ribonucleoside and FICA ribonucleoside were also metabolized extensively to their corresponding ribonucleoside 5′-triphosphates. 5′-Aminoimidazole-4-thiocarboxamide and 3-aminopyrazole-4-thiocarboxamide were metabolized only slightly to their ribonucleoside 5′-triphosphates. [³H]ribavirin was metabolized extensively to its 5′-triphosphate in L5178Y cells but was not detectably incorporated into RNA. Ribavirin caused a substantial decrease in the pool size of GTP in L5178Y cells and a concomitant increase in the pool size of both CTP and UTP.     

The effect of 2-ethyl-3-(4-hydroxybenzoyl)-benzofuran (benzarone) on the metabolism of arterial tissue and cultured arterial smooth muscle cells

Studies on the action of 2-ethyl-3-(4-hydroxybenzoyl)-benzofuran (benzarone) on the metabolism of cultured arterial smooth muscle cells and arterial tissue had the following results: 1. On incubation of calf arterial tissue in the presence of 0.03--0.1 mmol/l benzarone (8--26 micrograms/ml medium) the metabolic transformation of [14C]-glucose to [14C]-lactate and 14CO2 and the incorporation of 14C radioactivity into the total lipids is not significantly altered as compared with control values. In cultured human arterial smooth muscle cells 0.03 mmol/l benzarone stimulates the incorporation of [14C]-acetate and [3H]-palmitate into the cellular lipids while the receptor mediated uptake of homologous low-density lipoproteins (LDL) by the cells and their release are not influenced. 2. In concentrations greater than 0.2 mmol/l benzarone the glucose utilisation of arterial tissue is enhanced, while the labelling of lipids, in particular the labelling of the triglyceride fraction, is depressed. Under the same conditions the protein biosynthesis and the incorporation of [14C]-acetate and [3H]-palmitate into the total lipids of cultured arterial smooth muscle cells are decreased.     

IgE-immunotoxins. I. IgE-intact ricin

An IgE immunotoxin consisting of rat IgE myeloma protein, IR 162, conjugated via the heterobifunctional linking agent N-succinimidyl-3-(2-pyridyldithio)propionate to intact ricin was synthesized and evaluated. The capacity of this IgE-immunotoxin to bind to rat basophilic leukemia cells (RBL cells) and to inhibit RBL cell incorporation of [3H]leucine was assessed. The IgE-intact ricin conjugate sensitized RBL cells for histamine release after treatment with anti-IgE with a time-course of sensitization and dose-response equivalent to native IgE. Intact ricin and IgE-intact ricin were both cytotoxic to RBL cells as assessed by [3H]leucine incorporation. Lactose (50 mM) competed with intact ricin binding and toxicity such that more than 100 ng/ml ricin (8 times its IC50 in the absence of lactose) was required for ricin to kill RBL cells in the presence of lactose. Lactose (50 mM) was not able to fully inhibit 1-100 ng/ml IgE-ricin immunotoxin killing of RBL cells. Saturation of RBL cell IgE receptors by preincubation with IgE totally inhibited IgE-intact ricin-induced toxicity, in the presence of lactose, indicating that toxicity required IgE Fc receptor binding.     

The effect of the cytostatic agent 9-α-d-arabinofuranosyladenine on the nucleic acid metabolism in L5178Y cells

The influence of 9-α-d-arabinofuranosyladenine (α-araAdo) on cell metabolism of mouse lymphoma cells (L5178y) has been studied on cellular as well as on subcecllular level. α-AraAdo strongly inhibits the cell proliferation of L5178y cells. Starting with 3 × 10³ cells/ml and an incubation period of 72 hr, the drug reduces the cell proliferation to 50 per cent at a concentration of 12 μM. The cells die because of “unbalanced growth”. The cytostatic effect of α-araAdo can be abolished by coincubation with deoxyadenosine but not by adenosine. At cytostatic concentrations α-araAdo inhibits selectively the incorporation rate of thymidine into DNA but not the incorporation rates of precursors into RNA or protein. α-AraAdo is intracellularly phosphorylated to α-araATP. The compound has no effect on the extent of intracellular phosphorylation of exogenous administered adenosine or deoxyadenosine. α-AraAdo is incorporated into DNA but not into RNA; one molecule of α-araAdo is incorporated per 14,000 molecules of deoxyadenosine.     

Potentiation of CB 1954 cytotoxicity by reduced pyridine nucleotides in human tumour cells by stimulation of DT diaphorase activity.

The toxicity of CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] towards human cells was greatly enhanced by NADH (when foetal calf serum was present in the culture medium) and by nicotinamide riboside (reduced) (NRH), but not by nicotinate riboside (reduced). Co-treatment of human cells with CB 1954 and NADH resulted in the formation of crosslinks in their DNA. The toxicity produced by other DNA crosslinking agents was unaffected by reduced nicotinamide compounds. When caffeine was included in the medium, a reduction in the cytotoxicity of CB 1954 occurred. The toxicity experienced by human cell lines after exposure to CB 1954 and NADH was proportional to their levels of the enzyme DT diaphorase NAD(P)H dehydrogenase (quinone), EC 1.6.99.2. It is concluded that NRH, which we have shown to be a co-factor for rat DT diaphorase (Friedlos et al., Biochem Pharmacol 44: 25-31, 1992), is generated from NADH by enzymes in foetal calf serum, and stimulates the activity of human DT diaphorase towards CB 1954.     

Enhancement of pyrimidine nucleoside uptake into K562 and YAC-1 cells by cadeguomycin

Cadeguomycin markedly stimulated the uptake of thymidine, deoxycytidine and uridine into the acid-insoluble fraction of K562 human leukemic cells, but did not significantly affect adenosine incorporation. The enhancement of pyrimidine nucleoside uptake was 6 approximately 17 fold over the control. Aspartate incorporation into nucleic acid was not significantly blocked by the antibiotic, suggesting that the stimulation of pyrimidine nucleoside incorporation is not due to the inhibition of de novo pyrimidine nucleotide synthesis. Net DNA and RNA syntheses, observed by [32P]phosphate uptake, were not significantly affected by cadeguomycin. The enzymatic activity of thymidine, deoxycytidine and uridine kinases was higher in cadeguomycin-treated cells than in untreated cells, suggesting that the enhancement of pyrimidine nucleoside uptake occurs in the phosphorylation process. The stimulatory activity of cadeguomycin of thymidine uptake was reversed by guanosine and deoxyguanosine, but not by adenosine and deoxyadenosine, suggesting that intracellular metabolism and/or action of cadeguomycin is related to that of guanosine and deoxyguanosine. The stimulation of pyrimidine nucleoside incorporation by cadeguomycin was also found with YAC-1 cells, but not with the other cell lines. The enhancement effect of the antibiotic seems to be not directly related to its cytotoxicity.     

Synergistic effect of 5-fluorouracil and N-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in a human mammary carcinoma

The biological effects of $\text{N-(}\text{phosphonacetyl}\text{)-}\text{l}\text{-}\text{aspartate}$ (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10^?7 to 1.5 × 10^?5M) and PALA (6.0 × 10^?5 to 3.6 × 10^?3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [³H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [¹⁴C]deoxyguanosine and [³H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10^?5M PALA and 1 × 10^?6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10^?6M [³H]-5-FU into poly(A)RNA in comparison to [³H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10^?5 M PALA and 1 × 10^?6 M [³H]-5-FU resulted in a 285 per cent increase in the incorporation of [³H]-5-FU into non-poly(A)RNA. The incorporation of either [¹⁴C]deoxyguanosine or [³H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [³H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.     

Role of peroxidases, thiols and Bak/Bax in tumor cell susceptibility to Cu[DEDTC]2

Copper and two molecules of diethyl dithiocarbamate [DEDTC] form the Cu[DEDTC](2) complex, which shows cytotoxicity against melanoma and carcinoma cells, making it a potentially useful anti-cancer agent. The differential response to Cu[DEDTC](2) in susceptible human SKBR3 carcinoma and C8161 melanoma cell variants of moderate and high resistance to this organometallic complex was evaluated in this study. Both cell lines underwent apoptosis-associated PARP cleavage, changes in expression of nuclear NFkB p65, p21WAF1 and cyclin A, with loss of clonogenicity in response to this agent. However, a threefold greater concentration [IC(50) 0.6 microM DEDTC: 0.3 microM Cu] was required to kill moderately resistant C8161 melanoma compared to highly susceptible SKBR3 cells. Decreased susceptibility to Cu[DEDTC](2) in C8161 melanoma correlated with greater levels of glutathione peroxidase and catalase, and a fourfold lower requirement for N-acetyl cysteine (1mM) to overcome toxicity. Whereas melanoma cells selected for resistance to [0.8 microM DEDTC: 0.4 microM Cu] showed persistent catalase and GPx activity, melanoma cells with moderate susceptibility showed decreased catalase and Gpx when responding to treatment. Cytotoxic response in moderately susceptible C8161 melanoma cells involved an early accumulation of pro-apoptotic Bax in the G2 cell cycle phase, followed by an increased ratio of pro-apoptotic Bak to anti-apoptotic Mcl-1 in mitochondria. Our data suggests that Cu[DEDTC](2) toxicity is mediated through an increase in pro-apoptotic Bak/Bax via disruption of the peroxide and thiol metabolism.     

Characterization of covalently modified deoxyribonucleosides formed from dibenz[a,j]anthracene in primary cultures of mouse keratinocytes

Identification of various deoxyribonucleoside adducts formed in primary cultures of mouse keratinocytes exposed to dibenz[a,j]anthracene (DB[a,j]A) is presented. A preliminary analysis of the DNA adducts formed from 7-methyldibenz[a,j]anthracene (7MeDB[a,j]A) also is presented. Cultures of keratinocytes obtained from dorsal skins of female SENCAR mice were exposed to 0.5 microgram of tritium-labeled hydrocarbons/mL of medium for 24 h. The total DNA binding was 2.23 +/- 0.54 and 5.28 +/- 0.97 pmol of hydrocarbon/mg of DNA for DB[a,j]A and 7MeDB[a,j]A, respectively. These binding values represented the radioactivity associated with the modified deoxyribonucleosides separated from the normal deoxyribonucleosides on Sephadex LH-20 columns following enzymatic digestion of isolated DNA. Treatment of keratinocytes with DB[a,j]A produced adduct peaks corresponding to marker adducts derived from trans addition of both deoxyguanosine as well as deoxyadenosine residues to the (+) enantiomer of the anti-diol epoxide where the deoxyadenosine adducts were predominant. In addition, DNA adduct peaks corresponding to markers of trans and cis addition, respectively, of deoxyguanosine and deoxyadenosine to the (+)-syn-diol epoxide were also noted in these chromatograms. A major DNA adduct in cells exposed to DB[a,j]A was tentatively identified as resulting from the addition of deoxyadenosine to DB[a,j]A-5,6-oxide. Several other later eluting DNA adduct peaks, not corresponding to any of the marker adducts, were also present in these chromatograms. In comparison, when cells were exposed to the more biologically potent 7-methyl analogue, at least 12 DNA adduct peaks were consistently observed in HPLC chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)