Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

Inducibility of 6-thioguanine-resistant (6TGr) mutants and single-strand scission of DNA by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Frequency of 6TGr mutants increased concentration dependently by 24-h treatment with CdCl2 up to 3 X 10(-6) M but decreased beyond 3 X 10(-6) M. Mutagenic potency of cadmium in the absence of S9 was about half that of benzo[a]pyrene in the presence of S9 at equitoxic concentrations. Treatment of the cultured cells with cadmium after benzo[a]pyrene treatment was not synergistic but additive to the mutagenicity of benzo[a]pyrene. Single-strand scission of DNA by alkaline elution techniques was observed in the cells treated with CdCl2 for 2 h in a concentration-dependent manner. The single-strand scission by cadmium was detected only in combination with proteinase K digestion of the cell lysates, indicating formation of DNA--protein cross-linking by the metal. These biological and biochemical findings indicate that cadmium is mutagenic in mammalian cells, and its mutagenic effect seems to be accompanied by single-strand scission of DNA.     

Enhancing effects of cytosine arabinoside on ethyl methanesulfonate-induced 6-thioguanine resistance mutations in Chinese hamster V79 cells

We studied the synergistic enhancement effects of two chemicals which are different in their mechanism of action on DNA in cells. The test chemicals used were ethyl methanesulfonate (EMS) as an alkylating agent and cytosine arabinoside (Ara-C) as an analogue of cytidine. For determination of mutagenesis we measured the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells. EMS had a strong mutagenic effect on V79 cells, but for Ara-C the results were less clear. In this study, Ara-C had no detectable effect in inducing mutation up to a concentration of 5 X 10(-4) M. The mutation frequency of combined treatment with EMS and Ara-C was significantly higher than that obtained with EMS alone. These results indicate that Ara-C had an enhancing effect on mutations induced by EMS.     

DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67+/-19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations > or =100 microM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations > or 300 microM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by dl-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (> or =1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.     

Antimutagenic action of cobaltous chloride on radiation-induced mutations in cultured Chinese hamster cells

The effects of cobaltous chloride on 8-azaguanine (8AG)-resistant mutations induced by gamma-rays or ultraviolet (UV) light in cultured Chinese hamster V79 cells were examined. Cobaltous chloride alone had no significant effects on survival and mutations of V79 cells at concentrations less than 1 x 10(-5) M. Cobaltous chloride at a concentration of 3 x 10(-6) M had a marked effect in reducing 8AG-resistant mutations induced by gamma-rays of 2-6 Gy, when cells were incubated for 6-7 days in the presence of cobaltous chloride after gamma-ray irradiation (posttreatment). The pretreatment of cells with cobaltous chloride for 6 days before gamma-ray irradiation reduced 8AG-resistant mutations induced by gamma-rays. Pre- or post-treatment with cobaltous chloride had no such effect on UV-induced mutations, however. The difference in responsiveness to cobaltous chloride between bacterial and mammalian cell systems is discussed.     

Optimization of an assay for studying the effects of agents on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in washed human platelets

Before one can examine the effects of substances on the metabolism of arachidonic acid (AA) by the cyclooxygenase and lipoxygenase pathways, an assay system which allows one to detect increases or decreases in both pathways in needed. In order to develop such a system, we have examined nonaggregating washed human platelets (10(8) platelets/0.5 ml) incubated for various times with 2 microCi 3H-AA and increasing concentrations of AA. T/B2, HHT, 12-HETE, and AA were extracted and separated using reverse phase-HPLC. We first calculated the mass of AA products formed with 10(-7) to 10(-4) M AA and found that the cyclooxygenase was saturated with 10(-5) M AA whereas the lipoxygenase was not saturated with 10(-4) M AA. Cyclooxygenase products were more prevalent than 12-HETE below 10(-5) M AA, while lipoxygenase products predominated at 3 x 10(-5)-10(-4) M AA. Using 3 microM AA, which does not saturate the cyclooxygenase, we examined the effect of 0.25-10 minute incubation durations on the distribution of AA metabolites and found AA product formation to increase throughout this period without completely depleting the substrate. Since substrate depletion does not occur and further metabolism could be detected for both pathways with a 5 minute incubation with 3 microM AA, these incubation parameters were chosen in order to further test the assay system. Using these parameters, we found that 10(-4) M 5-hydroxytryptamine enhanced platelet 12-HETE formation and decreased T/B2 and HHT formation, thus demonstrating the capacity of this system to simultaneously detect changes in cyclooxygenase and lipoxygenase enzyme metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin production in cultured gastric mucosal cells: role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE2, PGI2 and thromboxaneA2 (TXA2) in amounts of 316 +/- 18, 100 +/- 7 and 30 +/- 5 pg per 10(5) cells in 10 min, respectively, in response to 10 microM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1-5 mM) did not alter the amount of subsequent AA-induced PGE2 production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE2 synthesis, while it increased intracellular cAMP accumulation. Our studies suggest AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, AA-induced PG production is limited in these cells, and it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67 ± 19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations ≥100 μM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations ≥300 μM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by DL-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (≥1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.     

Compartmentation of Cadmium and Iron in Mesembryanthemum crystallinum Plants during the Adaptation to Cadmium Stress

The common ice plants (Mesembryanthemum crystallinum) at the stage of five leaf pairs were exposed to cadmium chloride solutions (1, 0.1, and 0.01 mM) under the conditions of water culture. After five days, the partition of cadmium and iron in the plant organs and in the cell structures of the apical root region were investigated. Plant adaptation to excess cadmium in the environment was assessed by an increase in the leaf and root weight, a change in peroxidase activity, and an accumulation of proline. The common ice plant accumulated cadmium mainly in the root system. At a high concentration of cadmium in the nutrient solution (1 mM), its content in the root exceeded 2 g/kg fr wt, while at a concentration of 0.01 mM, it was as low as 10 mg/kg. Dithizone staining of transverse sections of the root apical region showed that, after a 48-h-long exposure of plants to 0.1 mM cadmium chloride, cadmium was localized in the cell walls of endodermis and metaxylem. The level of cadmium in leaves varied from 0.5 to 18 mg/kg fr wt. However, there was only a weak correlation between cadmium accumulation and the extent of a biomass decrease in the leaves of various stories, when cadmium concentration in the medium (1 mM cadmium chloride) was toxic. This fact could be related to a marked efflux of endogenous iron from old leaves into the young ones and to a change in the cadmium/iron ratio in the tissues. Proline accumulation in the third leaf pair and in the roots occurred at a relatively low cadmium content (10–12 mg/kg fr wt) in these organs. Maxima of activity of all three forms of peroxidase, viz., soluble, ionically-bound, and covalently-bound peroxidases, in roots were found at a high accumulation of cadmium in these organs (45 mg/kg fr wt). These maxima exceeded 3–4-fold the activity in aging leaves containing 5 mg cadmium/kg fr wt. A decrease in peroxidase activity in leaves was accompanied by a 3.3-fold decrease in iron content; thus, it could be caused by a deficiency of available iron necessary for the enzyme functioning. It was concluded that the resistance of Mesembryanthemum crystallinum, a halophyte, to excess cadmium content in the medium was achieved by its predominant accumulation in roots, where excess cadmium is compartmentalized in the apoplast and seems to be subjected to detoxification through pectate formation. Moreover, the leaves and, particularly, the roots are characterized by a high activity of the antioxidant systems, such as guaiacol-dependent peroxidases, and an occurrence of proline at modest cadmium concentrations.     

Interactive effects of cadmium and all-trans-retinoic acid on the induction of forelimb ectrodactyly in C57BL/6 mice

BACKGROUND: Most toxicological studies have tested single chemical agents at relatively high doses, and fewer studies have addressed the toxic effects of chemical interactions. It is important to understand the toxicity of chemical mixtures in order to assess the more realistic risks of environmental and occupational exposures. A number of chemicals are known to induce a predominantly postaxial forelimb ectrodactyly in C57BL/6 mice, including acetazolamide, ethanol, cadmium, valproic acid, carbon dioxide, dimethadione, phenytoin, and 13-cis-retinoic acid and all-trans-retinoic acid (RA). In the present study, the interactive effects of coadministration of cadmium and RA on developing limbs were investigated. METHODS: Pregnant C57BL/6 mice were treated with different intraperitoneal (IP) doses of cadmium chloride (CdCl2) and/or RA on gestational day (GD) 9.5, and fetuses were collected on GD 18 and double stained for examination of skeletal defects. RESULTS: When RA was given simultaneously with cadmium, a significant increase in the incidence and severity of forelimb ectrodactyly (predominantly postaxial) was observed compared to the results with corresponding doses of cadmium or RA alone. When mice were exposed to subthreshold doses of both cadmium (0.5 mg/kg) and RA (1 mg/kg), the combined treatment exceeded the threshold, resulting in forelimb ectrodactyly in 19% of the fetuses. Moreover, coadministration of cadmium and RA at doses exceeding the respective thresholds showed a synergistic effect, that is, 92% of fetuses were found with the forelimb defect as opposed to 10% if the response were additive. CONCLUSIONS: The findings demonstrate that concurrent exposure to these teratogens can have a synergistic effect and that subteratogenic doses may combine to exceed a threshold.     

A novel system combining biocatalytic dephosphorylation of L-ascorbic acid 2-phosphate and electrochemical oxidation of resulting ascorbic acid

An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from -0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2V, and reached to 5.0 μA cm(-2) at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction.     

Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules

Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.     

Protective effect of zinc on cadmium-induced micronuclei in V79 cells

The induction of micronuclei (MN) in mitotically active cells has been widely used and promoted as a biological marker of exposure to environmental toxins. In our study the effect of zinc on cadmium genotoxicity was investigated in V 79 cells. The results indicate that cadmium chloride exposure for 24 h increased micronucleus frequency and the percentage of binucleated cells in dose-dependent manner. At the highest concentration of cadmium (50 microM Cd) 23 MN were found in 1000 cells. The protective effect of zinc on cadmium genotoxicity was investigated at lower concentrations (5-25 microM CdCl2). At 50 microM Cd, the number of MN increased significantly (16 MN).     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Uterine arterial responses to arachidonate in pregnant and nonpregnant rabbits

Several investigators have suggested that prostaglandins (PG) may play a major regulatory role in maintaining uteroplacental blood flow in pregnancy. The present study was undertaken to assess the response of the uterine artery from near-term pregnant and nonpregnant rabbits to the PG precursor Na-arachidonate (AA) (C 20:4). Isolated uterine arterial strips were equilibrated isometrically under their optimal resting tensions in physiologic salt solution. Uterine arteries from pregnant rabbits elicited significantly greater contractile responses to arachidonate over the dose-range studied (10(-10)-10(-3) M) than did arteries from nonpregnant rabbits. These contractions were seen whether the strip was relaxed or precontracted with potassium chloride (30 mM). The contractile responses to AA were antagonized in a competitive manner by pretreating the arteries with the cyclooxygenase inhibitors meclofenamate (10(-5) M) or indomethacin (10(-5) M), thus suggesting that the contractile response to AA was the result of its conversion to prostanoids by the cyclooxygenase pathway. The possibility that the AA response was a general fatty acid effect was ruled out since oleate (C 18:1) had no effect on the arteries. In addition, prostaglandins F2 alpha and E2 (10(-5) M) also contracted the uterine arteries from the pregnant group. It is concluded from these studies that the uterine arterial wall from near-term pregnant rabbits utilizes the PG precursor, AA, for the production of prostanoids which, in turn, cause uterine arterial constriction.     

Mutagenicity of cadmium in Salmonella typhimurium and its synergism with two nitrosamines

Cadmium chloride (CdCl2) at concentrations of 0.5 mM was significantly mutagenic in Salmonella typhimurium tester strains and reverted histidine auxotrophy due either to missense (TA1975 and TA1535) or to frameshift (TA1537) mutations. It also induced forward mutations to 8-azaguanine resistance in each strain, but failed to increase mutation frequencies in strain TA100. More importantly, CdCl2 increased the mutagenicity of two common nitrosamines in synergistic fashion, at a level up to 30-fold greater than expected from simple additivity. The mutation frequency induced by N-methyl-N'-nitro-N-nitrosoguanidine was increased about 10-fold in the presence of 0.5 mM CdCl2. This synergism was seen both in the induction of 8-azaguanine resistance and the reversion of histidine auxotrophy and was observed in the repair-proficient strain TA1975 as well as its repair-defective (uvrB-) derived strain TA1535. The synergism was dependent upon Cd concentration and was much reduced at 0.25 mM CdCl2. The strongest synergism was observed in the reversion of histidine auxotrophy in TA1975 by 180 microM methylnitrosourea and 0.5 mM CdCl2. In contrast to mutagenicity, there was no evidence for synergism in the toxicity of CdCl2. These data suggest that cadmium might interfere with the repair of both spontaneous and nitrosamine-induced mutations. They also raise the possibility that cadmium and nitrosamines may have synergistic effects as environmental carcinogens.     

Viscum album prevents haematological changes,electrolyte imbalance,changes in liver function enzymes and histological alterations in some selected tissues in cadmium chloride-intoxicated rats

BackgroundThe use of Viscum album to treat different diseases is popular in the practise of alternative medicine. We investigated the ability of the aqueous extract of V. album to protect against the toxic effects of cadmium.MethodsThirty rats used for the experiment were treated as follows; Group 1 – no cadmium or extract. Group 2–10 mg/kg body weight of cadmium chloride. Group 3–10 mg/kg body weight of cadmium chloride and 200 mg/kg body weight of aqueous extract of V. album. Group 4–10 mg/kg body weight of cadmium chloride and 400 mg/kg body weight of aqueous extract of V. album. Group 5–10 mg/kg body weight of cadmium chloride with 800 mg/kg body weight of aqueous extract of V. album. Group 6–10 mg/kg body weight of cadmium chloride and atorvastatin (100 mg/kg body weight).ResultsApart from WBC and platelets, other haematological parameters and electrolytes, urea and creatinine levels were not significantly affected by the administration of cadmium chloride along with the aqueous extract of V. album. Treatment with the extract caused significant decreases in the hepatosomatic index, cardiosomatic index, and increase in renosomatic index of the test rats. It also resulted in significant (P < 0.05) decrease in AST level. Histological report also shows that treatment with the extract restored the normal myocardium and vascular architecture of the heart, normal portal and vascular architecture of the liver and normal glomerular and tubular architecture of the kidney, in the cadmium-intoxicated experimental rats.ConclusionV. album protects against the toxic effects of cadmium chloride.     

Characterization and partial purification of a chloride- and calcium-dependent glutamate-binding protein from rat brain

A glutamate-binding protein was solubilized from rat brain synaptic plasma membranes using sodium cholate. Its properties were characterized after addition of exogenous phospholipids and formation of proteoliposomes. Glutamate binding was dependent on calcium and chloride ions with maximal binding at concentrations of 10(-5) M calcium and 10 mM chloride ions. The effects of the two ions were synergistic rather than additive. In addition, glutamate binding was not affected by inhibitors specific for N-methyl-D-aspartate and kainate receptor subtypes, but was inhibited by quisqualate (Ki = 50 microM) and DL-2-amino-4-phosphonobutyrate (Ki = 1.3 mM). Furthermore, binding was abolished by 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1 mM dithiothreitol. These properties resemble those of the chloride- and calcium-dependent binding site. Starting from the detergent extract, the glutamate-binding protein was purified 123-fold using fractionated ammonium sulfate precipitation, chromatography on hydroxyapatite and on DEAE-Sephacel as sequential purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein fraction showed two major bands migrating with Mr values of 51,000 and 105,000. The properties of the partially purified binding protein were similar to those of the detergent extract. Glutamate binding to the partially purified protein is not due to a sequestration process or product binding to N-acetylated alpha-linked dipeptidase. Thus, the functional role of the binding protein remains to be established.     

Mechanism of the synergistic cytotoxicity between pentachlorophenol and copper-1,10-phenanthroline complex: the formation of a lipophilic ternary complex

When non- or sub-toxic levels of pentachlorophenol (PCP) and bis-(1, 10-phenanthroline)cupric complex, Cu(II)(OP)(2), were combined, a remarkable synergistic toxicity was observed as indicated by growth inhibition and bacterial inactivation. Similar synergistic cytotoxic effects were observed with other polychlorinated phenols and other positively charged cupric complexes. The synergism observed for these chemicals and similar reactive pairs of chemicals was found to be due to the formation of lipophilic ternary complexes which facilitated copper transport into the bacterial cells. The formation of ternary complexes of similar lipophilic character could be of relevance as a general mechanism of toxicity.     

DNA-damaging potential and glutathione depletion of 2-cyclohexene-1-one in mammalian cells, compared to food relevant 2-alkenals

2-Cyclohexene-1-one (CHX) occurs as a natural ingredient in some tropical fruits and has been detected as a contaminant in certain artificially sweetened soft drinks. To elucidate its cytotoxic/genotoxic effectiveness, CHX was tested in mammalian cell lines (V79 and Caco-2) and in primary human colon cells in comparison to structurally related 2-alkenals. Inhibition of cell growth (IC(50)) and cytotoxicity (LC(50)) were determined by protein staining with sulforhodamin B (SRB) and by trypan blue exclusion, respectively. DNA damage--both strand breaks and oxidised purines--was quantified by comet assay. Depletion of glutathione was measured in a kinetic assay, based on 5-thio-2-nitrobenzoate (TNB) formation. For CHX, a moderate cytotoxicity was observed after 1h incubation in V79 cells (LC(50): 4.75mM). The 2-alkenals ((E)-2-octenal (OCTE), (2E,4Z)-2,4-hexadienal (HEXDI), (E)-2-nonenal (NONE), (2E,6Z)-2,6-nonadienal (NONDI)) exhibited a distinctly higher cytotoxicity, except for (E)-2-hexenal (HEX) (LC(50): 3.67mM) and cinnamaldehyde (CA) (LC(50): 4.45mM). If the incubation time was prolonged to 24h, an IC(50) of 15microM was obtained for CHX which is well within the range obtained for the 2-alkenals (4 and 17microM). Concentration-dependent DNA damage was observed after 1h incubation with CHX. The respective DC(50) values (concentration inducing DNA damage in 50% of cells) were 272microM (V79) and 455microM (Caco-2). All 2-alkenals were more active under these conditions, except for CA. In primary human colon cells, CHX (800microM, 30min) exhibited a weak, but still significant DNA-damaging potential. Glutathione levels in V79 cells were effectively depleted (down to approximately 20%) by CHX concentrations not yet inducing DNA damage (c < or = 50microM). Incubation with CHX or 2-alkenals (50 and 100microM, 1h), followed by H2O2 treatment (5min, 25microM) resulted in increased levels of oxidised purines in the modified comet assay. CHX and HEX, additionally tested in primary human colon cells, depleted glutathione and increased the sensitivity towards oxidative stress.     

Ascorbic Acid and a Cytostatic Inhibitor of Glycolysis Synergistically Induce Apoptosis in Non-Small Cell Lung Cancer Cells

Ascorbic acid (AA) exhibits significant anticancer activity at pharmacologic doses achievable by parenteral administration that have minimal effects on normal cells. Thus, AA has potential uses as a chemotherapeutic agent alone or in combination with other therapeutics that specifically target cancer-cell metabolism. We compared the effects of AA and combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small cell lung cancer (NSCLC) cell lines to the effects on an immortalized lung epithelial cell line. AA concentrations of 0.5 to 5 mM caused a complete loss of viability in all NSCLC lines compared to a <10% loss of viability in the lung epithelial cell line. Combinations of AA and 3-PO synergistically enhanced cell death in all NSCLC cell lines at concentrations well below the IC₅₀ concentrations for each compound alone. A synergistic interaction was not observed in combination treatments of lung epithelial cells and combination treatments that caused a complete loss of viability in NSCLC cells had modest effects on normal lung cell viability and reactive oxygen species (ROS) levels. Combination treatments induced dramatically higher ROS levels compared to treatment with AA and 3-PO alone in NSCLC cells and combination-induced cell death was inhibited by addition of catalase to the medium. Analyses of DNA fragmentation, poly (ADP-ribose) polymerase cleavage, annexin V-binding, and caspase activity demonstrated that AA-induced cell death is caused via the activation of apoptosis and that the combination treatments caused a synergistic induction of apoptosis. These results demonstrate the effectiveness of AA against NSCLC cells and that combinations of AA with 3-PO synergistically induce apoptosis via a ROS-dependent mechanism. These results support further evaluation of pharmacologic concentrations of AA as an adjuvant treatment for NSCLC and that combination of AA with glycolysis inhibitors may be a promising therapy for the treatment of NSCLC.