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M Wiessler

Publications and source records attributed to M Wiessler.

At least 37 records · Page 2Linked to original sources
PubMed1999

Analysis of the inhibition of N-nitroso-dimethylamine activation in the liver by N-nitro-dimethylamine using a new non-linear statistical method.

N-nitro-dimethylamine (NTDMA) is carcinogenic to rats: it induces nasal cavity tumours. It can be demethylated to N-nitromethylamine and formaldehyde and reduced to N-nitroso-dimethylamine (NDMA): a potent liver carcinogen and also of the nasal cavity if activation in the liver is blocked. To explain the mechanism of NTDMA carcinogenicity we compared its demethylation with that of NDMA in liver microsomes from female and male rats, untreated, fasted or treated with ethanol to induce cytochrome P450 2E1 (CYP2E1). Kinetic parameters were analysed by nonlinear statistical methods, which yielded unbiased parameter estimates for the calculated Km and Vmax values. Km for both compounds was very similar in females (24-47 microM) whereas Vmax for NTDMA was consistently higher than for NDMA as substrate: 1.07-4.70 nmol formaldehyde/mg microsomal protein x min and 0.52-2.76 nmol, respectively. In liver microsomes from induced male rats NTDMA was found to be a much more effective inhibitor of NDMA activation (KEI 39.6-73.6 microM) than NDMA of NTDMA demethylation (KEI 224-286 microM). Nasal microsomes can demethylate both NDMA and NTDMA but the kinetics are vastly different. NTDMA is demethylated at a linear rate and approximately 10-fold more effectively than NDMA. The mechanism of carcinogenicity of ingested NTDMA, we propose, is a partial reduction to NDMA in the liver and inhibition of NDMA activation in the liver by residual NTDMA, which enables NDMA to reach the nasal mucosa where it is activated to DNA-alkylating species and the observed tumours are formed.

Animals↗
PubMed1998

Transport of the new chemotherapeutic agent beta-D-glucosylisophosphoramide mustard (D-19575) into tumor cells is mediated by the Na+-D-glucose cotransporter SAAT1.

For beta-D-glucosylisophosphoramide mustard (beta-D-Glc-IPM), a new alkylating drug in which isophosphoramide mustard is stabilized, a higher selectivity and lower myelotoxicity was observed than for the currently used cytostatic ifosfamide. Because beta-D-Glc-IPM is hydrophilic and does not diffuse passively through the lipid bilayer, we investigated whether a transporter may be involved in the cellular uptake. A variety of cloned Na+-sugar cotransporters were expressed in Xenopus oocytes, and uptake measurements were performed. By tracer uptake and electrical measurements it was found that beta-D-Glc-IPM was transported by the low-affinity Na+-D-glucose cotransporter SAAT1, which had been cloned from pig and is also expressed in humans. At membrane potentials between -50 and -150 mV, a 10-fold higher substrate affinity (Km approximately 0.25 mM) and a 10-fold lower Vmax value were estimated for beta-D-Glc-IPM transport than for the transport of D-glucose or methyl-alpha-D-glucopyranoside (AMG). Transport of beta-D-Glc-IPM and glucose by SAAT1 is apparently performed by the same mechanism because similar sodium dependence, dependence on membrane potential, electrogenicity, and phlorizin inhibition were determined for beta-D-Glc-IPM, D-glucose, and AMG. Transcription of human SAAT1 was demonstrated in various human carcinomas and tumor cell lines. In one of these, the human carcinoma cell line T84, phlorizin inhibitable uptake of beta-D-Glc-IPM was demonstrated with substrate saturation and an apparent Km of 0.4 mM. The data suggest that the Na+-D-glucose cotransporter SAAT1 transports beta-D-Glc-IPM into human tumor cells and may accumulate the drug in the cells. They provide an example for drug targeting by employing a plasma membrane transporter.

Animals↗
PubMed1998

Assessment of glucosylifosfamide mustard biodistribution in rats with prostate adenocarcinomas by means of in vivo 31P NMR and in vitro uptake experiments.

A combined in vitro/in vivo study was performed to evaluate the possible application of phosphorus (31P) NMR spectroscopy for therapy monitoring and to investigate glucosylifosfamide mustard (Glc-IPM) transport and biodistribution by radiotracer techniques. Dynamic in vivo 31P NMR measurements were performed in rats with prostate adenocarcinoma after i.v. injection of 1 mmol/kg body weight (bw) of ifosfamide (IFO) (n = 4) and 1 mmol/kg bw (n = 4) or 2.15 mmol/kg bw (n = 9) of Glc-IPM. In a biodistribution study with 14C-labeled Glc-IPM and a final dose of 0.8 mmol Glc-IPM/kg bw, the animals were killed 5, 30, 60, and 120 min after drug administration, an ethanol extraction was performed from several tissues, and the dose per g tissue was calculated. The same tumor cell line was used in saturation and competition experiments to further elucidate the transport mechanism. The 31P NMR signals of IFO and Glc-IPM showed no overlap with the endogenous phosphorus peaks. A rapid washout with a half-life between 25.9 +/- 5.6 min for the lower dose and 34.3 +/- 4.2 min for the higher dose of Glc-IPM was observed in the tumor. No statistically significant change of the pH value was observed during the examination period. The beta-nucleoside 5'-triphosphate (NTP)/inorganic phosphate (Pi) signal intensity ratio showed a tendency to decrease but without statistical significance. A rapid elimination was demonstrated by both the noninvasive NMR technique and the biodistribution study. No saturation was found in vitro for the Glc-IPM uptake, even at the concentration of 5 mM. Furthermore, the Glc-IPM uptake was not inhibited by the presence of 2-deoxyglucose and vice versa. The data show that the pharmacokinetics of Glc-IPM in the tumor can be followed in vivo by 31P NMR. The results presented are evidence for diffusion as the transport mechanism for Glc-IPM in this tumor model. However, the better visualization of Glc-IPM as compared to ifosfamide may be due to metabolic trapping of a negatively charged metabolite after deglycosylation.

Adenocarcinoma↗
PubMed1997

Chemopreventive effects of S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine against benzo[a]pyrene.

The putative antimutagenic/anticarcinogenic organosulfur compound, S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine (AC-DDTC), has been demonstrated to inhibit the metabolic activation and the genotoxicity of N-nitrosodiethylamine. We have investigated the chemopreventive activity of AC-DDTC against benzo[a]pyrene (B[a]P) in the Salmonella typhimurium bacterial mutation assay, in the chromosome aberration assay using Chinese hamster lung fibroblast (CHL), and in the mouse micronucleus assay in bone marrow cells. In the bacterial mutation assay, AC-DDTC produced a concentration dependent decrease in the number of mutant colonies induced by B[a]P. The chromosome damaging responses of B[a]P in CHL cells were abolished by the treatment of AC-DDTC, approximately to the level of the control. In the in vivo mouse bone marrow micronucleus test, pretreatment of AC-DDTC 1 h prior to B[a]P reduced the frequency of micronucleated polychromatic erythrocytes. The inhibitory effects were statistically significant and dose-dependent. Our results demonstrate that AC-DDTC, one of the mixed disulfide model compounds of disulfiram, prevents the mutagenic effects of B[a]P.

Acetylcysteine↗
PubMed1997

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases.

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.

Animals↗
PubMed1997

32P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy.

Recently, we reported that aristolochic acid (AA) a naturally occurring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the presence of additional AA-DNA adducts by the 32P-post-labelling method not only in the kidneys, but also in a ureter obtained after renal transplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), but also the minor adducts, 7-(deoxyguanosin-N2-yl)-aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relative adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of aristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed ingested the natural mixture of AAI and AAII. 32P-post-labelling analyses of further biopsy samples of one patient showed the known adduct pattern of AA exposure not only in the kidney, but also in the ureter, whereas in skin and muscle tissue no adduct spots were detectable. In an attempt to explain the higher level of the dA-AAI adduct compared to the dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contrast to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the kidney of rats. Our data demonstrate that AA forms DNA adducts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA observed in animals could also be responsible for the urothelial cancers observed in two of the CHN patients.

Adult↗
PubMed1996

Mixed disulfides from disulfiram inhibit the benzo[a]pyrene induced mutagenesis.

Some mixed disulfides derived from disulfiram and endogenous thiol compounds have been synthesized, biochemically characterized and their potential antigenotoxic effects have been proposed. The present study evaluated the mutagenic and antimutagenic specificities of two mixed disulfides using S. typhimurium reversion assay, namely S-(N,N-diethyldithiocarbamoyl)-N-acetylcysteine (AC-DDTC) and-L-glutathione (GS-DDTC). The two mixed disulfides were not mutagenic to Salmonella strains TA98 and TA100 in the presence or absence of S9 mixture. The increased number of revertants by benzo[a]pyrene (B(a)P) has been reduced to the control level by the preincubation with AC-DDTC or GS-DDTC. It was not due to the killing effect of B(a)P, mixed disulfides or B(a)P-disulfide mixture. The antimutagenic effect of AC-DDTC was more potent than that of GS-DDTC. These results indicate that AC-DDTC and GS-DDTC may have a role to play in reducing the risk of mutagenic effects of B(a)P.

Animals↗
PubMed1996

Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy.

A unique type of rapidly progressive renal fibrosis, designated Chinese herbs nephropathy (CHN), has been described in young Belgian women who had followed a slimming regimen including recently introduced Chinese herbs (Stephania tetrandra and Magnolia officinalis). Aristolochic acid (AA), a known nephrotoxin and carcinogen, was suspected as its causal factor. To substantiate this hypothesis, renal tissue from five patients with CHN and six patients with other renal diseases was analyzed for the presence of AA-derived DNA adducts, a described biomarker of AA exposure associated with its carcinogenic and mutagenic activity. Using the 32P-postlabeling method, a major distinct DNA adduct spot was found in all five cases of CHN and identified by cochromatographic analyses with authentic markers as the deoxyadenosine adduct of AA-I [7-(deoxyadenosin-N6-yl)-aristolactam I], the major component of the plant extract AA. This DNA adduct was absent in the six control cases. The 7-(deoxyadenosin-N6-yl)-aristolactam I adduct levels in CHN ranged from 0.7 to 5.3/10(7) nucleotides. Our data demonstrate that AA is implicated in CHN. They suggest a mechanism for the urothelial atypia and cancers observed in this disease and raise the possibility that a DNA mutation is responsible for the kidney-destructive fibrotic process.

Adult↗
PubMed1996

Pharmacokinetics and whole-body distribution of the new chemotherapeutic agent beta-D-glucosylisophosphoramide mustard and its effects on the incorporation of [methyl-3H]-thymidine in various tissues of the rat.

beta-D-Glucosylisophosphoramide mustard (beta-D-Glc-IPM) is a new, potential chemotherapeutic agent currently under investigation. Its pharmacokinetics in plasma and elimination of the parent drug and its metabolites via urine, bile, and exhaled air were studied in female Sprague-Dawley rats after bolus injection of 315 mg/kg. Typically, the drug's disposition from plasma follows a linear two-compartment model with half-lives (t1/2) of 1.8 (t1/2 alpha) and 32 min (t1/2 beta). The rate of clearance is 0.0046 (range 0.0030-0.0071) 1 min-1 kg-1, and the steady-state volume of distribution (Vss) is 0.18 (0.08-0.042) 1/kg (mean +/- inter-individual standard deviation). In human plasma, 28.1 +/- 2.6% (mean +/- SD) of the drug (concentration range 0.5-5 mg/ml) is bound to plasma proteins (predominantly to albumin). Biliary excretion of the parent drug accounts for 2.9 +/- 1.7% of the dose; its elimination in the form of 14CO2 via exhaled air is less than 1%. Within 24 h, 63.5 +/- 4.9% of the 14C-labeled drug is excreted unchanged in the urine, whereas 17.5 +/- 5.1% is excreted in the urine as metabolites. In addition, beta-D-Glc-[14C]-IPM was given as a bolus injection to female Sprague-Dawley rats at dose levels of 315 and 56.2 mg/kg. The distribution of radioactivity into tissue was examined qualitatively by whole-body autoradiography (WBA). Parallel experiments were carried out using the high dose of the L-derivative. After dosing with the D-compound, the highest levels of radioactivity were found in the liver, kidneys, thymus, thyroid gland, and central nervous system, including the brain. A similar distribution pattern was observed for the L-compound, except in the brain, which contained negligible levels of radioactivity. The distribution of the D-compound (high dose) was also investigated in male Copenhagen rats bearing a Dunning prostate tumor. The results were similar to those obtained in healthy Sprague-Dawley rats. Additionally, radioactivity was found in the tumor at 1 h after dosing with the drug and remained there even after 24 h. The effects of beta-D-Glc-IPM on the incorporation of [methyl-3H]-thymidine into the DNA of the liver, kidneys, thymus, spleen, esophagus, and bone marrow of the rat were examined following tissue excision and liquid scintillation counting at 2, 8, and 24 h after administration of the drug. beta-D-Glc-IPM showed no effect on the incorporation of [methyl-3H]-thymidine in the liver and an insignificant reduction in kidney DNA (maximal reduction: -27.3%). However, after 8 h there was a marked reduction in the incorporation rate in the thymus (-83.7%), spleen (-74.6%), and esophagus (-87.2%), with a tendency toward recovery within 24 h. In bone marrow cells a reduction of -75.5% (8 h) and -73.3% (24 h) was observed.

Animals↗
PubMed1995

Effect of site-specifically located aristolochic acid DNA adducts on in vitro DNA synthesis by human DNA polymerase alpha.

In order to examine the effect of purine adducts of the plant carcinogen aristolochic acid (AA) on DNA replication four 30-mer templates were prepared which contained single site-specific AA lesions. The oligonucleotides were isolated by HPLC and shown to contain the two known aristolochic acid I-DNA adducts (dA-AAI, dG-AAI) or the two known aristolochic acid II-DNA adducts (dA-AAII, dG-AAII) at position 27 from the 3' end by 32P-postlabeling. These adducts templates were replicated in primer (23-mer) extension reactions catalysed by human DNA polymerase alpha. Both AAI-DNA adducts (dA-AAI, dG-AAI) blocked DNA synthesis predominantly (80-95%) at the nucleotide 3' to the adduct, although primer extension to the full length of the template was found with unmodified control templates. Increasing dNTP concentrations had only a small effect on the DNA synthesis and translesional synthesis was negligible. In contrast, both AAII-DNA adducts showed marked differences in primer extension reactions. Blocking of DNA synthesis by the dA-AAII adduct was strongly dNTP dependent. With increasing dNTP concentrations 27 and 28 nucleotide products, indicating termination of DNA synthesis after incorporation of a nucleotide opposite this adduct and incorporation of an additional nucleotide accumulated. Only the dG-AAII adducted template allowed substantial translesional synthesis to the full length of the template (up to 25%). When a 26-mer primer was used to examine nucleotide incorporation directly across from the four purine adducts, we found no detectable incorporation of nucleotides for the dA-AAI adduct, whereas the dG-AAI adduct and both AAII-adducts (dA-AAII and dG-AAII) allowed preferential incorporation of the correct nucleotide. These results indicate that for human polymerase alpha three AA purine adducts (dA-AAI, dG-AAI and dA-AAII) provide severe blocks to DNA replication and that dG-AAII, which allows translesional synthesis, may not be a very efficient mutagenic lesion.

Aristolochic Acids↗
PubMed1995

D-19575--a sugar-linked isophosphoramide mustard derivative exploiting transmembrane glucose transport.

D-19575 is a glucose derivative of ifosfamide mustard with a broad spectrum of antitumor activity in animal models. In comparison with ifosfamide, D-19575 is less toxic and is better tolerated by tumor-bearing animals, achieving a better therapeutic efficacy. D-19575 is directly cytotoxic in vitro--in contrast to ifosfamide--and it is possible to modulate this cytotoxicity by inhibition of transmembrane glucose transporters. Correspondingly, renal reabsorption of filtered D-19575 could be blocked by pre- and cotreatment with phlorizin, resulting in a higher urinary excretion of the unchanged drug. The toxicity to white blood cells, colony-forming units (CFU-C), and spleen-cell colony-forming units (CFU-S) is considerably lower for D-19575 as compared with ifosfamide. In conclusion, D-19575 is a new alkylating cytotoxic agent with increased antitumor selectivity, probably caused by an active transmembrane transport mechanism.

Absorption↗
PubMed1995

Benzenediazonium ion derived from Sudan I forms an 8-(phenylazo)guanine adduct in DNA.

1-(Phenylazo)-2-hydroxynaphthalene (Sudan I, Solvent Yellow 14) is a liver and urinary bladder carcinogen in mammals. Sudan I forms benzenediazonium ion during cytochrome P-450 catalyzed metabolism. Calf thymus DNA was reacted with Sudan I activated by microsomal enzymes or with benzenediazonium ion in vitro, and the adducts formed were analyzed by the 32P-postlabeling technique. Both enrichment procedures (1-butanol extraction and nuclease P1 digestion) of this technique were employed for detection and quantitation of the DNA adducts formed. Cochromatographic analyses of adduct spots obtained by reaction with DNA or homopolydeoxyribonucleotides showed that the major Sudan I-DNA adduct was formed with deoxyguanosine. This adduct was also found in DNA directly reacted with benzenediazonium ion. The major Sudan I-DNA adduct was characterized by UV/vis absorbance spectroscopy as well as by the chromatographic properties of the adduct on cellulose or poly(ethylenimine)--cellulose TLC and HPLC. The characteristics are identical to those of the adduct synthesized from benzenediazonium ion and guanine, identified by mass, UV/vis, and 1H-NMR spectroscopy as 8-(phenylazo)guanine. The results suggest strongly that benzenediazonium ion derived from Sudan I reacts with DNA in vitro to form the stable 8-(phenylazo)guanine adduct.

Adenine↗
PubMed1995

Inhibitory effects of mixed disulfides from disulfiram on the metabolism and genotoxicity of N-nitrosodiethylamine.

Disulfiram (CAS 97-77-8, DSF), a potent anticarcinogenic compound, is known to form mixed disulfides with sulfhydryl group containing amino acids or proteins in vivo. In the present study the stabilities of two mixed disulfides which may arise in the metabolism of disulfiram, i.e. S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine (AC-DDTC) and S-(N,N-diethyldithiocarbamoyl)-L-glutathione (GS-DDTC) in phosphate buffer (pH 7.2) and in rat liver subcellular fractions were investigated as well as their influences on the glutathione (GSH)-related detoxifying system, on the metabolism of [14C] N-nitrosodiethylamine (NDEA) and on the genotoxic activity of NDEA in rats. Both substances were stable in buffer and in microsomes but were degraded in cytosol showing a half life of 4.7 h (AC-DDTC) and 3.2 h (GS-DDTC). Addition of GSH to the incubation media accelerated the degradation of mixed disulfides in cytosol. In vivo administration of AC-DDTC and GS-DDTC (1.7 mmol/kg i.p.) led to an increase in hepatic GSH content and to an inhibition of the activity of NDEA deethylase. Both mixed disulfides inhibited the metabolism of NDEA. After a 28 mg/kg i.p. dose of [14C] NDEA only 0.4% was excreted unchanged in the urine. Pretreatment with AC-DDTC and GS-DDTC caused a 10 to 20 fold increase in the amount of NDEA excreted in the urine. The occurrence of DNA single strand breaks in rat liver cells induced by NDEA was completely neutralized by the pretreatment with AC-DDTC.

Animals↗
PubMed1995

Fate and distribution of [14C]S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine, an antimutagenic mixed disulfide from disulfiram, in rats.

S-(N,N-Diethyldithiocarbamoyl)-N-acetyl-L-cysteine (AC-DDTC) is a mixed disulfide from disulfiram and N-acetyl-L-cysteine, which possesses putative anticarcinogenic and antimutagenic properties. The present study describes the absorption, distribution, metabolism and excretion of 14C-labeled AC-DDTC in rats. AC-DDTC was well absorbed after oral administration. Based on the excretion of radioactivity in urine, the minimum absorption was about 73%. The rate of absorption was very rapid, with the peak level of radioactivity in plasma after 15 min of administration. Mean Cmax value for N,N-diethyldithiocarbamate (DDTC) after oral dose of AC-DDTC (20 mg/kg) was 3.8 +/- 0.2 nmol/ml at 15 min and the mean residence time was 47.1 +/- 2.8 min. After oral administration of [14C]AC-DDTC, radioactivity was distributed relatively rapidly. Maximum concentrations were observed in the liver (0.443% dose/g), kidneys (0.496% dose/g), oesophagus (0.313% dose/g) and in the adrenals (0.364% dose/g) at 30 min to 1 h after dosing. Liver was the only organ which contains a considerable amount of radioactivity (0.091% dose/g) 24 h after dosing. Two metabolites of AC-DDTC following oral administration were identified in the plasma and liver by GC and HPLC using extractive alkylation technique, namely DDTC and its methyl ester. Urinary excretion was a major route of elimination of radioactivity derived from [14C]AC-DDTC, in that about 73% of the dose was recovered in urine whereas only 14% was found in feces over 7 days.

Acetylcysteine↗
PubMed1994

Sugar-linked dithiocarbamates as modulators of metabolic and genotoxic properties of N-nitroso compounds.

A series of putative anticarcinogenic and antimutagenic compounds was synthesized on the basis of tetraethylthiuram disulfide (disulfiram) and its metabolite, diethyldithiocarbamate (DDTC). Diallyldithiocarbamate was synthesized in order to combine the anticarcinogenic properties of diallyl sulfide, a known inhibitor of chemical carcinogenesis from Allium species, and those of DDTC. Several sugar-linked dithiocarbamates (SDTCs) were prepared using glucose, cellobiose, and lactose as glycosyl donors and DDTC and diallyldithiocarbamate as acceptors. All the S--glycoside bonds of SDTCs were very stable under physiological conditions in vitro. At low nitrosamine concentrations, glucose-DDTC inhibited microsomal nitrosamine dealkylases in vitro. In vivo these enzymes were also inhibited 4 h after i.p. administration of glucose-DDTC or lactose-DDTC to rats (1.7 mmol/kg); after 24 h, the values had returned to control levels. Glucose-DDTC induced the activity of glutathione-related enzymes. Concomitant treatment of rats with glucose-DDTC and N-nitrosodiethylamine (NDEA) led to a depression of the oxidative metabolism of [14C]NDEA to 14CO2 but increased the elimination of unchanged [14C]NDEA in the urine. Furthermore, glucose-DDTC totally inhibited the formation of DNA single-strand breaks induced by NDEA. All these effects may contribute to possible antimutagenic and anticarcinogenic actions of the dithiocarbamates investigated.

Animals↗
PubMed1994

Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid.

Synthetic oligonucleotides (18-mers) containing either a single deoxyadenosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main components of the plant carcinogen aristolochic acid (AA). These reactions resulted in the formation of site-specifically adducted oligonucleotides containing the two known AAI-DNA adducts (dA-AAI, dG-AAI) or the two known AAII-DNA adducts (dA-AAII, dG-AAII) at position 15 from the 3' end. Using HPLC chromatography, the oligonucleotides were purified and subsequently shown to contain the adducts of interest by 32P-postlabelling. The adducted oligonucleotides were used as templates in primer (11-mer) extension reactions catalysed by modified bacteriophage T7 DNA polymerase (Sequenase). Regardless of the type of DNA adduct examined, DNA synthesis was blocked predominantly (80-90%) at the nucleotide 3' to each adduct, although primer extension to the full length of the template was noted with unmodified control templates. However, 15 nucleotide products, indicating blocking of DNA synthesis after incorporation of a nucleotide opposite the adduct and translesional synthesis products were formed in all cases in different amounts, depending on the adduct structure. When a 14-mer primer together with high dNTP concentrations was used to examine nucleotide incorporation directly across from the four different purine adducts we found that the deoxyadenosine adducts (dA-AAI and dA-AAII) allowed incorporation of dAMP and dTMP equally well, whereas the deoxyguanosine adducts (dG-AAI and dG-AAII) allowed preferential incorporation of dCMP. Molecular dynamic simulations showed that the aristolactam moiety of all adducts exhibit a strong stacking, with the adenine residue at the 3' end of the 14-mer primer. These studies demonstrate that all AA purine adducts provide severe blocks to DNA replication and that the guanine adducts may not be very efficient mutagenic lesions. In contrast, the translesional bypass past adenine adducts of the aristolochic acids suggests a mutagenic potential resulting from dAMP incorporation by polymerase. AT-->TA transversion mutations would be the mutagenic consequences of AA adenine adducts, which are consistent with the activating mutations of c-ras genes found in AA-induced tumours of rodents.

Aristolochic Acids↗
PubMed1994

Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by 32P-postlabelling analysis using different chromatographic procedures.

We report the analysis of DNA adducts in the target organ (forestomach) of male Sprague-Dawley rats treated orally with two doses (10 mg/kg body wt) per week for 2 weeks of either aristolochic acid I (AAI), aristolochic acid II (AAII) or the plant extract aristolochic acid (AA). DNA adducts were detected and quantitated using the nuclease P1-enhanced version of the 32P-postlabelling assay. For identification of adducts, reference compounds were prepared by reaction of enzymatically activated AAI and AAII with 3'-purine phosphonucleosides and analysed by the n-butanol enrichment procedure. These reference compounds were assigned to the previously characterized DNA adducts of AAI [7-(deoxyguanosin-N2-yl)-aristolactam I = dG-AAI, 7-(deoxyadenosin-N6-yl)-aristolactam I = dA-AAI] and AAII [7-(deoxyadenosin-N6-yl)-aristolactam II = dA-AAII]. Cross referencing of the carcinogen-modified nucleoside bisphosphates obtained from forestomach DNA with the synthetic standard compounds by ion-exchange chromatography and reversed-phase HPLC demonstrated that the major DNA adducts formed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise, forestomach DNA isolated from AAII-treated rats showed two purine-derived adduct spots, the major one being dA-AAII, the minor one being tentatively identified as 7-(deoxyguanosin-N2-yl)-aristolactam II. A minor adduct detected in forestomach DNA of rats treated with AAI was found to be chromatographically indistinguishable from the adduct identified as dA-AAII, indicating a possible demethoxylation reaction of AAI. Quantitation of DNA adducts revealed that in in vitro reactions with 3'-phosphonucleosides the adduct levels were approximately one order higher for both AAI- and AAII-derived adducts than in forestomach DNA modified with AAI or AAII in vivo. In vitro as well as in vivo adduction by AAI was more efficient than adduction by AAII. The pattern of adduct spots obtained from forestomach DNA of rats treated with the plant extract AA reflected the composition of the extract determined by HPLC analysis. Irrespective of the aristolochic acid used to induce DNA adducts, deoxyadenosine is the major target of modification, pointing to the general importance of deoxyadenosine adducts for chemical carcinogenesis of these naturally occurring products. This study shows that the combination of two independent chromatographic systems considerably enhances the fidelity of identification of DNA adducts with the 32P-postlabelling assay.

Animals↗