Evaluation of a new model system for studying the formation of heterocyclic amines

Heterocyclic amines (HAs) are an important class of food mutagens and carcinogens, which can be found in cooked meat and fish. Increasing heating temperatures and times usually increase mutagenic activity in meat and meat extracts during cooking. We developed a model system, which allows to examine the effects of precursor composition and heating conditions (time and temperature) on the formation of HAs in meat. Homogenized and freeze dried meat samples (beef, pork chops, chicken breast and turkey breast) are heated with diethylene glycol in closed vials under stirring in a thermostated heating block. After an appropriate sample preparation (extraction and clean-up) ten different HAs were measured by HPLC analyses with gradient elution and mass selective detection. The time courses of HA-formation in the different kinds of meat at varying heating temperatures were determined up to heating times of 30 min. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was the most abundant HA in these experiments and reached the highest concentrations in the beef meat samples, as did the other HAs (MeIQ, AalphaC) at 220 degrees C in the heating block under stirred conditions. Additionally the influence of the antioxidant TBHQ (t-butylhydroquinone) on the formation of HAs in the model system was tested. However TBHQ effected only slight reductions of HA formation in all kinds of meat.     

Effect of intestinal microfloras from vegetarians and meat eaters on the genotoxicity of 2-amino-3-methylimidazo[4,5-f]quinoline, a carcinogenic heterocyclic amine

Aim of this study was to investigate the impact of intestinal microfloras from vegetarians and non-vegetarians on the DNA-damaging activity of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), a carcinogenic heterocyclic amine that is found in fried meats. Floras from four vegetarians (Seventh Day Adventists) and from four individuals who consumed high amounts of meats were collected and inoculated into germfree F344 rats. The rats were kept on isocaloric diets that either contained animal derived protein and fat (meat consumers group) or proteins and fat of plant origin (vegetarian groups). IQ (90 mg/kg bw) was administered orally, after 4 h the extent of DNA-damage in colon and liver cells was determined in single cell gel electrophoresis assays. In all groups, the IQ induced DNA-migration was in the liver substantially higher than in the colon. In animals harbouring floras of vegetarians, the extent of damage was in both organs significantly (69.2% in the liver, P<0.016 and 64.7%, P<0.042 in the colon, respectively) lower than in the meat consumer groups. Our findings show that diet related differences in the microfloras have a strong impact on the genotoxic effects of IQ and suggest that heterocyclic amines are less genotoxic and carcinogenic in individuals that consume mainly plant derived foods.     

Effects of garden and water cress juices and their constituents,benzyl and phenethyl isothiocyanates,towards benzo(a)pyrene-induced DNA damage: a model study with the single cell gel electrophoresis/Hep G2 assay

The aim of this study was to investigate the chemoprotective effects of water and garden cress juices towards benzo(a)pyrene (B(a)P)-induced DNA damage using the single cell gel electrophoresis (SCGE)/Hep G2 test system. This experimental model combines the advantages of the SCGE assay with that of human derived cells possessing inducible phase I and phase II enzymes. Treatment of Hep G2 cells with small amounts of water cress or garden cress juice (0.1-1.25 microl/ml) and B(a)P reduced the genotoxic effect of the latter in a dose-dependent manner. Contrary to the results with the juices, unexpected synergistic effects were observed with benzyl isothiocyanate (BITC, 0.6 microM), a breakdown product of glucotropaeolin contained abundantly in garden cress. Although these concentrations of BITC did not cause DNA damage per se, at higher concentrations (> or = 2.5 microM), the compound caused a pronounced dose-dependent DNA damage by itself. With phenethyl isothiocyanate (PEITC), the breakdown product of gluconasturtin contained in water cress, no synergistic effects with B(a)P were seen; however, significant induction of DNA damage was observed when the cells were exposed to the pure compound at concentrations > or = 5 microM. In experiments with (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE, 5.0 microM), the ultimate genotoxic metabolite of B(a)P, and the juices, only moderate protective effects were seen indicating that detoxification of BPDE is not the main mechanism behind the protective effect of the juices against B(a)P-induced DNA damage. In conclusion, our findings show that garden and water cress juices are highly protective against B(a)P-induced DNA damage in human derived cells and that their effects can not be explained by their isothiocyanate contents.     

DNA adduct formation of 14 heterocyclic aromatic amines in mouse tissue after oral administration and characterization of the DNA adduct formed by 2-amino-9H-pyrido[2,3-b]indole (AalphaC), analysed by 32P_HPLC.

Heterocyclic aromatic amines (HAAs) are produced during cooking of proteinaceous food such as meat and fish. Humans eating a normal diet are regularly exposed to these food-borne substances. HAAs have proved to be carcinogenic in animals and to induce early lesions in the development of cancer. DNA adduct levels in mouse liver have been measured by 32P-HPLC after oral administration each of 14 different HAAs. The highest DNA adduct levels were detected for 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), respectively. To assess a relative risk in a human population, a relative risk index was calculated by combining the DNA adduct levels in mouse liver with human daily intake of heterocyclic amines in a US and in a Swedish population. Such calculations suggest that AalphaC presents the highest risk for humans, e.g. nine-fold higher compared with the most abundant amines in food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP). Therefore, the distribution of DNA adducts in different tissues of mouse was investigated after oral administration of AalphaC. The highest AalphaC-DNA adduct levels were found in liver (137 adducts/10(8) normal nucleotides) followed by heart, kidney, lung, large intestine, small intestine, stomach and spleen, in descending order. To characterize the chemical structure of the major DNA adduct, chemical synthesis was performed. The major DNA adduct from the in vivo experiments was characterized by five different methods. On the basis of these results, the adduct was characterized as N2-(deoxyguanin-8-yl)-2-amino-9H-pyrido [2,3-b]indole. Considering the abundance of AalphaC not only in grilled meat, but also in other products like grilled chicken, vegetables and cigarette smoke and in light of the results of the present study, it is suggested that the human cancer risk for AalphaC might be underestimated.     

Occurrence of heterocyclic amines in several home-cooked meat dishes of the Spanish diet

Heterocyclic amines (HAs) were determined in several of the most frequently eaten meat dishes in Spain such as fried beef hamburger, fried pork loin, fried chicken breast, fried pork sausages, griddled chicken breast, griddled lamb steak and griddled beef steak. All of the products tested were household cooked. The HAs were analysed in the selected meat dishes using an analytical method based on solid-phase extraction followed by liquid chromatography coupled to tandem mass spectrometry. DMIP, MeIQx, 4,8-DiMeIQx, Norharman, Harman, PhIP, Trp-P-1, AalphaC and MeAalphaC were the amines most frequently found at concentrations of up to 47 ng g(-1) of cooked meat. Glu-P-2, IQ, MeIQ, Glu-P-1, 7,8-DiMeIQx and Trp-P-2 were only found in a few of the meat dishes and their concentrations were lower than 1 ng g(-1) of cooked meat. The highest amounts of HAs, especially PhIP and DMIP, were formed in fried chicken breast and the lowest were formed in fried beef hamburger and in fried pork sausages. Daily intake of HAs in Spain was estimated at 606 ng of mutagenic HAs per capita and day, DMIP and PhIP being the main contributors.     

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3'-azido-3'-deoxythymidine

This study tried to clarify the question if nuclear genotoxicity played a role in 3'-azido-3'-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH>13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.     

Hydroquinone-induced genotoxicity and oxidative DNA damage in HepG2 cells

Hydroquinone (HQ) is used as an antioxidant in rubber industry and as a developing agent in photography. HQ is also an intermediate in the manufacture of rubber, food antioxidant and monomer inhibitor. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of HQ and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. DNA strand breaks and DNA-protein crosslinks (DPC) were measured by the proteinase K-modified alkaline single cell gel electrophoresis (SCGE) assays. Using the SCGE assay, a significant dose-dependent increment in DNA migration was detected at concentrations of HQ (6.25-25 microM); but at the higher tested concentrations (50 microM), a reduction in the migration compared to the maximum migration at 25 microM was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of HQ (50 microM). A significant increase of the frequency of micronuclei was found in the range from 12.5 to 50 microM in the micronucleus test (MNT). The data suggested that HQ caused DNA strand breaks, DPC and chromosome breaks. To elucidate the oxidative DNA damage mechanism, the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were chosen to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH), respectively. The present study showed that HQ induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 25-50 and 6.25-50 microM, respectively. Moreover, HQ significantly caused 8-hydroxydeoxyguanosine (8-OHdG) formation in HepG2 cells at concentrations from 12.5 to 50 microM. All these results demonstrate that HQ exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress. GSH, as a main intracellular antioxidant, is responsible for cellular defense against HQ-induced DNA damage.     

Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A

Dietary iron may contribute to colon cancer risk via production of reactive oxygen species (ROS). The aim of the study was to determine whether physiological ferric/ferrous iron induces oxidative DNA damage in human colon cells. Therefore, differentiated human colon tumour cells (HT29 clone 19A) were incubated with ferric-nitrilotriacetate (Fe-NTA) or with haemoglobin and DNA breaks and oxidised bases were determined by microgelelectrophoresis. The effects of Fe-NTA were measured with additional H(2)O(2) (75microM) and quercetin (25-100microM) treatment. Analytic detection of iron in cell cultures, treated with 250microM Fe-NTA for 15 min to 24h, showed that 48.02+/-5.14 to 68.31+/-2.11% were rapidly absorbed and then detectable in the cellular fraction. Fe-NTA (250-1000microM) induced DNA breaks and oxidised bases, which were enhanced by subsequent H(2)O(2) exposure. Simultaneous incubation of HT29 clone 19A cells with Fe-NTA and H(2)O(2) for 15 min, 37 degrees C did not change the effect of H(2)O(2) alone. The impact of Fe-NTA and H(2)O(2)-induced oxidative damage is reduced by the antioxidant quercetin (75-67% of H(2)O(2)-control). Haemoglobin was as effective as Fe-NTA in inducing DNA damage. From these results we can conclude that iron is taken up by human colon cells and participates in the induction of oxidative DNA damage. Thus, iron or its capacity to catalyse ROS-formation, is an important colon cancer risk factor. Inhibition of damage by quercetin reflects the potential of antioxidative compounds to influence this risk factor. Quantitative data on the genotoxic impact of ferrous iron (e.g. from red meat) relative to the concentrations of antioxidants (from plant foods) in the gut are now needed to determine the optimal balance of food intake that will reduce exposure to this type of colon cancer risk factor.     

Hemoglobin and hemin induce DNA damage in human colon tumor cells HT29 clone 19A and in primary human colonocytes

Epidemiological findings have indicated that red meat increases the likelihood of colorectal cancer. Aim of this study was to investigate whether hemoglobin, or its prosthetic group heme, in red meat, is a genotoxic risk factor for cancer. Human colon tumor cells (HT29 clone 19A) and primary colonocytes were incubated with hemoglobin/hemin and DNA damage was investigated using the comet assay. Cell number, membrane damage, and metabolic activity were measured as parameters of cytotoxicity in both cell types. Effects on cell growth were determined using HT29 clone 19A cells. HT29 clone 19A cells were also used to explore possible pro-oxidative effects of hydrogen peroxide (H2O2) and antigenotoxic effects of the radical scavenger dimethyl sulfoxide (DMSO). Additionally we determined in HT29 clone 19A cells intracellular iron levels after incubation with hemoglobin/hemin. We found that hemoglobin increased DNA damage in primary cells (> or =10 microM) and in HT29 clone 19A cells (> or =250 microM). Hemin was genotoxic in both cell types (500-1000 microM) with concomitant cytotoxicity, detected as membrane damage. In both cell types, hemoglobin and hemin (> or =100 microM) impaired metabolic activity. The growth of HT29 clone 19A cells was reduced by 50 microM hemoglobin and 10 microM hemin, indicating cytotoxicity at genotoxic concentrations. Hemoglobin or hemin did not enhance the genotoxic activity of H2O2 in HT29 clone 19A cells. On the contrary, DMSO reduced the genotoxicity of hemoglobin, which indicated that free radicals were scavenged by DMSO. Intracellular iron increased in hemoglobin/hemin treated HT29 clone 19A cells, reflecting a 40-50% iron uptake for each compound. In conclusion, our studies show that hemoglobin is genotoxic in human colon cells, and that this is associated with free radical mechanisms and with cytotoxicity, especially for hemin. Thus, hemoglobin/hemin, whether available from red meat or from bowel bleeding, may pose genotoxic and cytotoxic risks to human colon cells, both of which contribute to initiation and progression of colorectal carcinogenesis.     

Genotoxic risk and oxidative DNA damage in HepG2 cells exposed to perfluorooctanoic acid

Perfluorooctanoic acid (C8HF15O2, PFOA) is widely used in various industrial fields for decades and it is environmentally bioaccumulative. PFOA is known as a potent hepatocarcinogen in rodents. But it is not yet clear whether it is also carcinogenic in humans, and the genotoxic effects of PFOA on human cells have not yet been examined. In this study, the genotoxic potential of PFOA was investigated in human hepatoma HepG2 cells in culture using single cell gel electrophoresis (SCGE) assay and micronucleus (MN) assay. In order to clarify the underlying mechanism(s) we measured the intracellular generation of reactive oxygen species (ROS) using dichlorofluorescein diacetate as a fluorochrome. The level of oxidative DNA damage was evaluated by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) in PFOA-treated HepG2 cells. PFOA at 50-400 microM caused DNA strand breaks and at 100-400 microM MN in HepG2 cells both in a dose-dependent manner. Significantly increased levels of ROS and 8-OHdG were observed in these cells. We conclude that PFOA exerts genotoxic effects on HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS.     

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.     

Palmitate-induced apoptosis in cardiomyocytes is mediated through alterations in mitochondria: prevention by cyclosporin A

Palmitate, a C16 fatty acid found in high concentrations in the blood in acute myocardial infarction, induces apoptotic cell death. To more completely define the nature and mechanism underlying palmitate-induced cell death, cardiomyocytes were cultured from embryonic chick heart and were treated with palmitate. Concentration-dependent loss of cell viability was established by loss of the ability of palmitate-treated cells to exclude propidium iodide (PI), metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and retain fluorescein diacetate (FDA). Dual staining with PI and FDA and subsequent analysis by FACS established that palmitate-induced cell death was predominantly necrosis whereas apoptosis occurred in 13% of all dead cells. The low proportion of palmitate-induced apoptosis was confirmed by evaluation of the DNA content or PI fluorescent staining of the DNA of permeabilized cardiomyocytes. A critical role for mitochondria in the pathogenesis of palmitate-induced cell death was demonstrated, for the first time, based on palmitate-induced reduction of mitochondrial activity as assessed by the mitochondrial-selective dye chloromethyl-X-Rosamine and the presence of a greater amount of the mitochondrial marker cytochrome C in the cytosol of palmitate-treated cardiomyocytes than in control cells. Further, cyclosporin that inhibits the development of mitochondrial transition pores blocked palmitate-induced alteration in mitochondrial function and palmitate-induced cell death. We further demonstrated the selectivity of cyclosporin A for the prevention of apoptotic cell death in the heart as there was no alteration in necrotic cell death produced by palmitate with cyclosporin pretreatment. Our data demonstrate the nature of palmitate-induced cell death in cardiomyocytes (both apoptotic and necrotic), propose a mitochondrial basis for its pathogenesis and show that cyclosporin A prevents palmitate-induced apoptotic cardiomyocyte cell death.     

Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis

The aim of the present study was to evaluate the induced genotoxicity (DNA damage) due to organophosphate pesticide profenofos (PFF) in gill cells of freshwater fish Channa punctatus using single cell gel electrophoresis (SCGE)/Comet assay. The 96h LC(50) value of PFF (50% EC) was estimated for the fish species in a semistatic system and then three sub-lethal of LC(50) concentrations viz the sub-lethal 1, sub-lethal 2 and sub-lethal 3 concentrations were determined as 0.58ppb, 1.16ppb and 1.74ppb, respectively. The fish specimens were exposed to these concentrations of the pesticide and the gill tissue samplings were done on 24h, 48h, 72h and 96h post exposure for assessment of DNA damage in terms of percentage of DNA in comet tails. In general, a concentration dependent response was observed in the gill cells with induction of maximum DNA damage at the highest concentration of PFF. The results of the present investigation indicated that PFF could potentially induce genotoxic effect in fish, even in sub-lethal concentrations and SCGE as a sensitive and reliable tool for in vivo assessment of DNA damage caused by the genotoxic agents.     

Single-cell gel electrophoresis assays with human-derived hepatoma (Hep G2) cells.

The purpose of the present study was the development of a protocol for detecting chemically-induced DNA damage, using the alkaline single-cell gel electrophoresis (SCGE) assay with human-derived, metabolically competent hepatoma (Hep G2) cells. Previous studies indicated that Hep G2 cells have retained the activities of certain phase I and phase II enzymes and reflect the metabolism of genotoxins in mammals better than other in vitro models which require addition of exogenous activation mixtures. The optimal trypsin concentration for the removal of the cells from the plates were found to be 0.1%. Dimethylsulfoxide, at concentrations up to 2%, was an appropriate solvent for water-insoluble compounds. To determine the optimal exposure periods for mutagen treatment, the time kinetics of comet formation was investigated with genotoxic chemicals representing various classes of promutagens namely benzo[a]pyrene (B[a]P), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and N-nitrosodimethylamine (NDMA) and with N-nitrosomethylurea (NMU). All compounds caused a statistically significant induction in DNA damage. With the promutagens, comet formation increased gradually as a function of the exposure duration, and reached maximum values between 20-24 h. With NMU, comet induction maximized already after a short exposure (1 h) and remained at a constant level for up to 24 h. Based on these results, the Hep G2/SCGE assay appears to be a suitable approach for investigating DNA damaging potential of chemicals. Further experiments with IQ and B[a]P showed that the assays are highly reproducible. Comparisons of the present results with those from earlier experiments in which other endpoints (induction of sister chromatid exchanges, micronuclei and chromosomal aberrations) were measured in Hep G2 cells, indicated that the sensitivity of the SCGE assays is more or less identical. Since the SCGE assay is less time consuming than other genotoxicity assays we anticipate that it might be a suitable approach to investigate DNA damaging effects of chemicals in the human-derived, metabolically competent cell line.     

Specificity of growth inhibition of melanoma by 4-hydroxyanisole

An experimental study using human melanoma (NEL-MI), rat hepatoma (Fu5-5), and human kidney (293-31) cell lines was undertaken in order to evaluate the antitumor activity of 4-hydroxyanisole (4-OHA) in vitro. Prior reports have indicated highly specific antitumor activity of 4-OHA against melanoma cells in vitro. This specific antitumor activity has been proposed to be due to the oxidation of 4-OHA by tyrosinase to cytotoxic oxidation products. Dose-dependent cytotoxicity was observed when cells were cultured for 72 h in the presence of 4-OHA. At 100 microM, 4-OHA produced growth inhibition of 62%, 32%, and 55% in melanoma, hepatoma, and kidney cell lines, respectively. No effect was seen at 10 microM 4-OHA. 1,000 microM 4-OHA produced 100% kill. Tyrosinase activity was detected only in melanoma cells. The effect of 100 microM 4-OHA on the incorporation of 3H DNA precursors in melanoma, hepatoma, and kidney cells was also studied. Thymidine incorporation was inhibited in all three cell lines at the lowest cell density tested, with the greatest inhibition seen on melanoma cells. As cell density increased, the effect of 4-OHA on thymidine incorporation decreased. With respect to RNA synthesis, 4-OHA significantly reduced the incorporation of uridine in all three cell lines, with the greatest effect in melanoma cells. Cell density also affected the inhibition of uridine incorporation, but to a lesser extent than that observed on thymidine incorporation. The effect of 4-OHA on leucine incorporation was modest and uninfluenced by cell density. Thus, cytotoxicity of 4-OHA may involve two different mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elucidation of strict structural requirements of brefeldin A as an inducer of differentiation and apoptosis

Brefeldin A (BFA) can induce a wide variety of human cancer cells to differentiation and apoptosis and is in development as an anticancer agent. To elucidate structural requirements for cytotoxicity and induction of differentiation and apoptosis, BFA was structurally modified into various derivatives including 4-epi-BFA in this study. Their inducing activities of apoptosis were evaluated with their cytotoxicities, DNA fragmentation and morphological changes in human colon cancer cell HCT 116. The cytotoxicity of 4-epi-BFA (TX-1923) (IC50 = 60 microM) was 300 times lower than that of BFA (IC50 = 0.2 microM). Furthermore, 4-epi-BFA induced DNA fragmentation and apoptotic morphological changes at much higher concentrations (70 and 50 microM, respectively) than BFA (0.11 and 0.36 microM, respectively). These results indicated that the configuration of 4-hydroxyl group of brefeldin A plays a key role in the cytotoxicity and induction of apoptosis. On the other hand, 7-O-acetyl-BFA, 4-O-acetyl-BFA, and 4,7-di-O-acetyl-BFA exhibited potential activities in cytotoxicity and inducibility of apoptosis. We suggested that the structural determinants for BFA include the moiety of the Michael acceptor, the conformational rigidity of the 13-membered ring, and the configuration of 4-hydroxyl group.     

Humic acids reduce the genotoxicity of mitomycin C in the human lymphoblastoid cell line TK6

The antimutagenic/desmutagenic activity of a leonardite humic acid (LHA) and a soil humic acid (SHA) was studied in the cultured human lymphoblastoid cell line TK6 treated with mitomycin C (MMC) as reference mutagen by evaluating the induction of micronuclei (MN). Two different concentrations of HA were used, 2.5 and 10 microg/ml, in three different treatments: (1) HA alone (genotoxic test); (2) HA after 2-h pre-incubation with 0.3 microM of MMC (desmutagenic test) and (3) combinations of HA and MMC at 0.3 microM without pre-incubation (antimutagenic test). Neither of the HA used alone did produce genotoxic effects, but both HAs reduced significantly the frequencies of MN induced by MMC, especially in the desmutagenic test. A slight cell-protective effect against the cytotoxicity of MMC was also exhibited by the two HAs in the desmutagenic test. The LHA showed a desmutagenic/antimutagenic activity that was more pronounced than that of SHA, which is possibly related to the higher carboxylic group content and lower phenolic group content of LHA. These results confirm the antigenotoxic action exerted by HAs in human cells, similarly to what has been previously observed in various plant species.     

Determination of NG,NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry

A fully validated gas chromatographic-mass spectrometric (GC-MS) method for the accurate and precise quantification of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography-chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for NG,NG-[2H6]-dimethyl-L-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of L-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 microM. The method was validated in a concentration range of 0-1.2 microM in cell culture medium and 0-2 microM in 50 microl aliquots of human plasma. The precision was > or =97% and the accuracy was determined to be > or =94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.     

人肝癌细胞p53基因的表达及其对细小病毒H-1的敏感性

本文利用单链构象多态性分析,17号染色体短臂等位基因杂合性分析,Northern印迹,免疫沉淀,p53基因第7外显子酶切等技术检测了两个中国人肝癌细胞系SMMC-7721,YY-8103和一个自发转化的人肝细胞系L-02的p53基因结构与表达。实验表明,这三个细胞系中没有出现17号染色体短臂等位基因杂合性缺失,第4—9外显子也没发生突变,但其mRNA和蛋白表达水平很低。利用MTT比色分析法研究了这三个细胞系和其他已知p53基因背景的八个人肝癌细胞系(QGY-7703、PLC/PRF/5、Huh-7、Hep3B、FOCUS、Tong/ HCC、SK-Hep-1、HepG2)对自主性细小病毒H-1的敏感性。除HepG2细胞外,其他十个细胞系p53基因的结构和/或表达都不正常。经H-1感染(moi=20)后,其敏感性均高于HepG2细胞。本研究初步表明了p53基因结构或表达的不正常可能导致人肝癌或转化细胞对H-1的敏感性的提高。     

Synthesis of [(18)O(2)]valproic acid and its use as an internal standard for the quantitative measurement by gas chromatography-electron ionization mass spectrometry

A specific method for the quantitative determination of valproic acid in human plasma is presented. Valproate was extracted from acidified plasma by hexane extraction and converted to its trimethylsilyl derivative without sample concentration. The derivatives were analyzed without any further purification. Using gas chromatography-electron ionization mass spectrometry, diagnostic useful fragment ions at m/z 201 and 205 were obtained for valproic acid and [(18)O(2)]valproic acid internal standard, respectively. [(18)O(2)]Valproic acid was synthesized from unlabeled valproate by acid-catalyzed exchange reaction in H(2)(18)O. The method was validated in the expected concentration range of a pharmacokinetic study. Thus, calibration graphs were linear within a range of 0.47-120 microgram/ml plasma. Intra-day precision was 2.29% (0.47 microgram/ml), 2.93% (4 microgram/ml), 3.22% (20 microgram/ml) and 4.40% (80 microgram/ml), inter-day variability was found to be 1.49% (0.47 microgram/ml), 3.79% (20 microgram/ml), 2.74% (40 microgram/ml) and 3.03% (80 microgram/ml). Inter-day accuracy showed deviations of 1.94% (0.47 microgram/ml), 0.53% (4 microgram/ml), -0.32% (20 microgram/ml) and 0.06% (80 microgram/ml). The method is rugged and robust and has been applied to the batch analysis of valproate during pharmacokinetic profiling of the drug.