DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the P-postlabeling technique

Using the ³²P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with β-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by ³²P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C₁₁---C₁₂ bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.     

Study of the inhibition of alpha-amylase by the benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine

Inhibition of porcine pancreas and human saliva alpha-amylase (EC 3.2.1.1) by sanguinarine and chelerythrine was studied. The inhibition of alpha-amylase was assayed using a biosensor method which utilises a flow system equipped with a peroxide electrode. 250 microM sanguinarine and 250 microM chelerythrine cause complete inhibition of 1.9 nkat alpha-amylase from porcine pancreas. The same concentration of sanguinarine and chelerythrine caused 23.9% and 7.5% inhibition, respectively, of 1.9 nkat alpha-amylase from human saliva. Mixed type and partially reversible inhibition was found for both alpha-amylases treated with either alkaloid.     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

32P-Postlabeling high-performance liquid chromatographic improvements to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene,and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene

Single compounds can generate complex DNA adduct patterns by reactions through different pathways, with different target nucleotides and through different configurations of the products. DNA adduct analysis by ³²P-HPLC was improved by adding an isocratic plateau in an otherwise linear gradient, thereby enhancing resolution of predictable retention time intervals. This enhanced ³²P-HPLC technique was used to analyze and at least partly resolve 14 out of 16 available benzo[c]phenanthrene deoxyadenosine and deoxyguanosine adduct standards, 8 out of 8 available benzo[a]pyrene deoxyadenosine and deoxyguanosine adduct standards, and 51 peaks from 7,12-dimethylbenz[a]anthracene-calf thymus DNA reaction products. The same type of gradient modifications could be used to enhance resolution in analyses of other complex DNA adduct mixtures, e.g., in vivo in humans.     

Quaternary benzo[c]phenanthridine alkaloids--novel cell permeant and red fluorescing DNA probes.

BACKGROUND: Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs-macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells. METHODS: Water stock solutions of the alkaloids (10-100 microg/ml) were added to intact cells, and after a brief incubation the cells were observed. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma), and LEP (human lung fibroblasts), and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analyzed by fluorescence microscopy and by flow cytometry. RESULTS: All tested alkaloids immediately entered living cells with MA, CHR, and SA binding to DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR, and SA stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. QBAs were excitable using common argon lasers (488 nm) emitting at a range of 575-755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. CONCLUSIONS: It was shown that MA, CHR, and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes, both in fluorescence microscopy and flow cytometry, including multiparameter analysis of peripheral blood and bone marrow. MA binds DNA stochiometrically and can provide information on DNA content.     

The benzo[c]phenanthridine alkaloid, sanguinarine, is a selective, cell-active inhibitor of mitogen-activated protein kinase phosphatase-1

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress. Small molecule inhibitors of MKP-1 have not been reported, in part because of the lack of structural guidance for inhibitor design and definitive assays for MKP-1 inhibition in intact cells. Herein we have exploited a high content chemical complementation assay to analyze a diverse collection of pure natural products for cellular MKP-1 inhibition. Using two-dimensional Kolmogorov-Smirnov statistics, we identified sanguinarine, a plant alkaloid with known antibiotic and antitumor activity but no primary cellular target, as a potent and selective inhibitor of MKP-1. Sanguinarine inhibited cellular MKP-1 with an IC50 of 10 microM and showed selectivity for MKP-1 over MKP-3. Sanguinarine also inhibited MKP-1 and the MKP-1 like phosphatase, MKP-L, in vitro with IC50 values of 17.3 and 12.5 microM, respectively, and showed 5-10-fold selectivity for MKP-3 and MKP-1 over VH-1-related phosphatase, Cdc25B2, or protein-tyrosine phosphatase 1B. In a human tumor cell line with high MKP-1 levels, sanguinarine caused enhanced ERK and JNK/SAPK phosphorylation. A close congener of sanguinarine, chelerythrine, also inhibited MKP-1 in vitro and in whole cells, and activated ERK and JNK/SAPK. In contrast, sanguinarine analogs lacking the benzophenanthridine scaffold did not inhibit MKP-1 in vitro or in cells nor did they cause ERK or JNK/SAPK phosphorylation. These data illustrate the utility of a chemical complementation assay linked with multiparameter high content cellular screening.     

Quaternary benzo[c]phenanthridine alkaloids as inhibitors of dipeptidyl peptidase IV-like activity baring enzymes in human blood plasma and glioma cell lines

Quaternary benzo[c]phenanthridine alkaloids (QBA), fagaronine (FA), sanguinarine (SA), chelerythrine (CHE) and the QBA extract from Macleya cordata (EX) exerted differential inhibitory effect on the hydrolytic activity of particular dipeptidyl peptidase (DPP)-like enzyme isolated from human blood plasma and from human and rat glioma cell lines. The low-MW form of DPP-IV-like enzyme activity, corresponding most probably to DPP-8, observed only in glioma cells but not in human plasma, was inhibited preferentially by SA, CHE and EX, and only slightly by FA. The alkaloid inhibitory effect was concentration-dependent in the range 25-150 mM and directly pH-related. In addition, a subtle but consistent inhibition of the intermediate-MW form of DPP-IV-like enzyme activity, ascribed to DPP-IV/CD26, observed only in human plasma and of the attractin (high-MW form of DPP-IV-like enzyme activity, expressed in U87 glioma cells) by the studied alkaloids was observed. We conclude that some of the QBA biological effects could be determined by tissue and cell type specific dipeptidyl peptidase IV-like molecules expression pattern.     

DNA adduct formation in human hepatoma cells treated with 3-nitrobenzanthrone: analysis by the (32)P-postlabeling method

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a (32)P-postlabeling/thin layer chromatography (TLC) method and a (32)P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36-36.4 microM) for 3h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone-3'-phosphate (dA3'p-N(6)-C2-ABA) and 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-N(2)-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3'p-N(6)-C2-ABA and its relative adduct labeling (RAL) value at 36.4 microM of 3-NBA was 200.8+/-86.1/10(8)nucleotide.     

The correlation between benzo[a]pyrene-induced mutagenicity and DNA adduct formation in Salmonella typhimurium TA100

In an attempt to stabilize the dose response in the Salmonella typhimurium test (STT), the use of DNA-bound products from BP was evaluated as a measure of the biologically effective dose. In addition to the previously documented interlaboratory variation, we observed a 3-fold difference in the dose response of TA100 to BP even when the assay was repeated with the same experimental conditions. When overall BP-DNA adduct formation was related to the level of His+ revertants, a series of responses emerged with two predominating. In the first type of response around 70 revertants per plate were generated for every BP molecule bound per 10(6) nucleotides of cellular DNA. The second response gave about 1400 revertants per plate for one BP bound in every 10(6) nucleotides. Several intermediates curves were also detected. The variation in the mutational response to binding levels occurred regardless of the source of S9 or the growth stage of the cells. These experiments indicate that there was no constant level of DNA damage that would lead to a specified number of revertants of TA100 by BP and that DNA modification was not solely responsible for mutagenic potency. It is possible that an induction of an error-prone repair function of the muc gene carried by the plasmid pKM101 in TA100 may be affecting the relationship between the measured adduct level and reversion frequency.     

Identification and characterization of a novel benzo[a]pyrene-derived DNA adduct

The aim of this study was to generate and identify a novel benzo[a]pyrene (BP)-derived DNA adduct found both in vitro and in vivo. To date, the majority of studies have focused on N(2)-[10 beta(7 beta,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]-deoxyguanosine (anti-BPDE-dG), the major adduct generated following bioactivation of BP. However, a second adduct is also formed following bioactivation of BP which has been speculated to result from further metabolism of 9-OH-BP. In order to identify this second reaction pathway, the ultimate DNA binding species, and the DNA base involved, we have synthesized and characterized a dG-derived DNA adduct arising from further bioactivation of 9-OH-BP in the presence of rat liver microsomes. Analysis of the adducted nucleotides was conducted using both the (32)P-postlabeling assay and capillary electrophoresis-mass spectrometry (CE-MS).     

Role of activated oxygen species in benzo[a]pyrene:DNA adduct formation in vitro.

The role of several activated oxygen species in the oxidation and binding of B[a]P to calf thymus DNA in vitro was investigated. B[a]P was reacted with calf thymus DNA in the presence and absence of scavengers of active oxygen species. Reactions were performed in the dark at 37 degrees C for 30 min in a buffered aqueous solution with 250 micrograms of calf thymus DNA. The levels of B[a]P:DNA adducts formed were determined using the 32P-postlabeling assay. B[a]P:DNA adduct levels ranged from 1.5-2.6 and 0.25 pmol adducts/mg DNA in reactions with 120 or 12 nmol of B[a]P, respectively. The addition of scavengers of reactive oxygen species to reaction mixtures resulted in a considerable decrease in the levels of DNA adducts formed in comparison to control reactions. Reactions performed with 500 units catalase or 100 units superoxide dismutase significantly inhibited DNA adduct formation. In these reactions adduct levels were 32 and 48% of control levels, respectively. The addition of both catalase and superoxide dismutase to reactions inhibited adduct formation by 95% relative to control reactions. A decrease in adduct levels was also observed when reactions were performed with citrate-Fe3+ chelate, a scavenger of superoxide. In reactions with 50 mM mannitol and 50 mM sodium benzoate, both of which are hydroxyl radical scavengers, adduct formation was significantly inhibited with adduct levels being 30 and 51% of control values, respectively. Adduct levels were decreased to 26% of control values in reactions with 10 mM 2,5-dimethylfuran, a scavenger of singlet oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA adduct formation in northern pike (Esox lucius) exposed to a mixture of benzo[a]pyrene, benzo[k]fluoranthene and 7H-dibenzo[c, g]carbazole: time-course and dose-response studies

The time-course and dose dependent formation of DNA adducts in juvenile northern pike (Esox lucius) following a single exposure to a mixture of benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF) and 7H-dibenzo[c,g]carbazole (DBC) were investigated by use of the (32)P-postlabelling assay. A complex adduct pattern was detected in liver and intestine of exposed fish. For the time-course studies fish were exposed either by oral administration or by intraperitoneal (i.p.) injection. Following a single i.p. injection of the mixture (40micromole/kg body weight of each substance) significantly elevated DNA adduct levels were detected in the liver after 1 day. Adduct levels were higher in liver than in intestine, in which significant elevation were detected from day 3 to 12. Following exposure via food (80micromole/kg body weight of each substance), adduct levels were detected in both liver and intestine 1 day after exposure, and continued to increase until day 3 in liver and day 6 in intestine. Calculation of a binding index, which compensates for differences in dosage, resulted in much higher adduct formation (five times in liver and 22 times in intestine) following oral exposure. Pikes receiving single oral doses of 12.5, 50, 100 or 200micromole/kg body weight of each substance exhibited significantly higher adduct levels in both liver and intestine compared to controls. Hepatic adduct levels were also higher in fish given 100 and 200micromole/kg compared to 12.5micromole/kg. Results from this study show that DNA adducts are rapidly formed in juvenile northern pike following both i.p. injection and feeding of a mixture of BaP, BkF and DBC. A maximum level was reached within a few days, which then persisted at approximately the same level for at least 9-12 days. The results also shows that higher levels of adducts were obtained following oral administration compared to i.p. injection, particularly in the intestine.     

Relative bioavailability and DNA adduct formation of benzo[a]pyrene and metabolites in the diet of the winter flounder

• 1.1. Radiolabeled metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) were shown to be absorbed through the diet of the winter flounder, Pseudo pleuronectes americanus.
• 2.2. Oral bioavailability of a mixture of naturally produced metabolites was significantly less than that of the parent BaP.
• 3.3. Oral bioavailability of a pure metabolite, BaP-7,8-dihydrodiol (7,8-D) was found to be similar to that of BaP.
• 4.4. Both metabolites and BaP formed DNA adducts in liver.

     

DNA adduct formation and persistence in liver and extrahepatic tissues of northern pike (Esox lucius) following oral exposure to benzo[a]pyrene, benzo[k]fluoranthene and 7H-dibenzo[c,g]carbazole.

The formation and persistence of DNA adducts in liver, intestinal mucosa, gills and brain of juvenile northern pike (Esox lucius) following oral exposure to benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF) and 7H-dibenzo[c,g]carbazol (DBC) were analysed by 32P-postlabelling. The dosage was 25 micromol/kg body weight of each substance, administered on 5 occasions with an interval of 12-14 days. Sampling was carried out 9 days after the second treatment, and 9, 16, 33 and 78 days after the fifth treatment. Pikes were also fed with the substances singly for comparison of adduct patterns. A complex pattern of adducts was detected in all examined tissues from fish treated with the mixture. Total adduct levels were highest in intestine (347+/-17.4 nmol adducts/mol nucleotides, mean+/-SE), followed by liver (110+/-9.3), gills (69+/-6) and brain (14+/-4.2). In pike treated with BaP alone, one major adduct was detected in all examined tissues. This BaP-adduct made up approximately 50% of the total amount of adducts in the brain. Corresponding values in liver, intestine and gills were 23, 31 and 34%, respectively. One relatively weak BkF-adduct and at least 10 different DBC-adducts were detected in all analysed tissues. Total adduct level in the intestine declined to 29.4% of the maximum value 78 days after the last exposure, while there was no significant decline in adduct levels in liver, gills or brain. The results suggest that intestine is more susceptible to adduct formation than liver after oral exposure, and that adduct levels in the intestine represent ongoing or relatively recent exposure. DNA adducts in the other investigated tissues were much more persistent and may therefore accumulate during long-term exposure.     

Relative bioavailability and DNA adduct formation of benzo[a]pyrene and metabolites in the diet of the winter flounder.

1. Radiolabeled metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) were shown to be absorbed through the diet of the winter flounder, Pseudopleuronectes americanus. 2. Oral bioavailability of a mixture of naturally produced metabolites was significantly less than that of the parent BaP. 3. Oral bioavailability of a pure metabolite, BaP-7,8-dihydrodiol (7,8-D) was found to be similar to that of BaP. 4. Both metabolites and BaP formed DNA adducts in liver.     

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase -- (32)P-postlabeling analysis of DNA adduct formation

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the (32)P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (alpha-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.     

DNA damage induced by cigarette smoke condensate in vitro as assayed by 32P-postlabeling. Comparison with cigarette smoke-associated DNA adduct profiles in vivo.

Cigarette smoke induces a multitude of bulky/aromatic DNA adducts in vivo as revealed by 32P-postlabeling assay. The formation of such adducts is thought to involve metabolic activation of aromatic chemicals especially polycyclic aromatic hydrocarbons (PAHs) present in tumor-initiating cigarette tar fractions, via cytochrome P450-associated monooxygenases. Because radicals are present in both the gas and particulate (tar) phase of cigarette smoke and in aqueous extracts of cigarette smoke condensate (CSC), we addressed the question as to whether cytochrome P450-independent, possibly free radical-mediated reactions may contribute, also, to formation of cigarette smoke-associated bulky DNA adducts. Rat-lung DNA was incubated with aqueous extracts of CSC in the absence of microsomes under various conditions and analyzed by 32P-postlabeling. Radioactively labeled bulky reaction products were found to accumulate in a time- and CSC concentration-dependent manner. The resulting chromatographic profiles resembled cigarette smoke-associated DNA-adduct patterns observed in vivo. Pretreatment of aqueous CSC extract with radical scavengers/reducing agents (ascorbic acid, glutathione) diminished adduct formation in a concentration-dependent manner. Adduct formation in vitro may involve oxygen-free radicals, which are known to be present in aqueous CSC extracts and could (i) attack DNA directly to produce bulky adducts, (ii) induce radical sites on DNA covalently binding CSC components, or (iii) convert CSC components to DNA-reactive electrophiles. In addition, DNA may react with direct-acting mutagens in CSC. Adduct fractions derived from in vitro and in vivo experiments showed similar chromatographic behavior, suggesting that metabolic activation as well as processes not involving metabolism lead to formation of smoking-induced bulky DNA adducts in vivo.     

DNA adduct formation in human hepatoma cells treated with 3-nitrobenzanthrone: analysis by the P-postlabeling method

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a ³²P-postlabeling/thin layer chromatography (TLC) method and a ³²P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36–36.4 μM) for 3 h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2′-deoxyadenosin-N⁶-yl)-3-aminobenzanthrone-3′-phosphate (dA3′p-N⁶-C2-ABA) and 2-(2′-deoxyguanosin-N²-yl)-3-aminobenzanthrone-3′-phosphate (dG3′p-N²-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3′p-N⁶-C2-ABA and its relative adduct labeling (RAL) value at 36.4 μM of 3-NBA was 200.8 ± 86.1/10⁸ nucleotide.     

Identification of benzo[c]phenanthridine metabolites in human hepatocytes by liquid chromatography with electrospray ion-trap and quadrupole time-of-flight mass spectrometry

The metabolism of the benzo[c]phenanthridine alkaloids was studied using human hepatocytes which are an excellent model system for biotransformation studies. For analysis of the alkaloids and their metabolites, an electrospray quadrupole ion-trap mass spectrometry (ESI ion-trap MS) connected to a reversed phase chromatographic system based on cyanopropyl modified silica was used. The optimized experimental protocol allowed simultaneous analysis of the alkaloids and their metabolites and enabled study of their uptake into and interconversion in human hepatocytes. The results show that formation of the dihydro metabolite which may be followed by specific O-demethylenation/O-demethylation processes, is probably the main route of biotransformation (detoxification) of the benzo[c]phenanthridines in human hepatocytes. The structure of the main O-demethyl metabolite (2-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-c]phenanthridin-1-ol; 336.1 m/z,) was proposed by the multi-stage MS and quadrupole time-of-flight MS methods using chemically synthesized standard.