Barbara Bertram

Induction of poly(ADP-ribosyl)ation and DNA damage in human peripheral lymphocytes after treatment with (-)-epigallocatechin-gallate.

With regard to a future use of tea polyphenols in intervention trials with individuals at high cancer risk, the effects of the tea ingredient (-)-epigallocatechin gallate (EGCG) on poly(ADP-ribose) (PAR) levels and on DNA damage were investigated in human lymphocytes. A dose- and time-dependent elevation of both PAR formation as assessed by quantitative immunofluorescence analysis and DNA damage as assessed by the comet assay were observed after treatment with EGCG at 20, 40 and 80 microM for 10-240 min. Maximum levels of PAR formation and of DNA damage were observed after 10 min at all concentrations tested. Increased PAR levels were still detectable by 240 min in the 40 and 80 microM groups. At the lowest concentration, which is near the physiological peak values found after tea ingestion, PAR formation was not correlated with DNA damage. Here, EGCG led to pronounced PAR levels, whereas the comet assay was almost negative. In contrast, such marked differences in time course and extent of both genotoxicity and PAR formation following EGCG treatment were not detected after gamma-irradiation. Our results suggest that the known chemopreventive effects of EGCG, the main constituent of tea, may be partly attributed to an induction of PAR formation.

Reduced glutathione inhibits the alkylation by n-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.

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

Abstract β-D-Glucosylisophosphoramide mustard (β-DGlc-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 (t1/2α) and 32 min (t1/2β). The rate of clearance is 0.0046 (range 0.0030–0.0071) l min-1 kg-1, and the steady-state volume of distribution (Vss) is 0.18 (0.08–0.042) l/kg (mean±interindividual 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, β-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 β-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. β-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.

Effects of disulfiram on mixed function oxidase system and trace element concentration in the liver of rats

Disulfiram (DSF), an inhibitor of chemically induced carcinogenesis, and its metabolite diethyldithiocarbamate (DDTC) have been investigated for their influence on trace element distribution and on certain enzymes of the drug metabolizing system in the livers of phenobarbital (PB) treated rats. Both substances diminished the PB induced enzyme response in liver microsomes, DDTC being more effective (-85%) than DSF (-60%). The copper, cobalt and zinc content of the livers of DSF treated animals were increased by factors of 6, 3 and 1.5 respectively as compared to controls, while DDTC treatment had no influence on liver trace element content. A correlation between enzyme inhibition and enhanced trace element uptake of the liver after DSF administration could not be observed. The change of trace element transport into the liver during DSF treatment is discussed.

Influence of a prolonged treatment with disulfiram andd(-)penicillamine on nitrosodiethylamine-induced biological and biochemical effects in rats

SummaryThe influence of a prolonged treatment with disulfiram (DSF) andd(-)penicillamine (PA) on biological and biochemical effects induced by nitrosodiethylamine (NDEA) was studied in rats. The combination of NDEA and DSF led to a massive and early development of esophageal tumors, which were fatal to the animals. No liver tumors were observed in this group, whereas PA in combination with NDEA led to an increased development of liver tumors compared with NDEA alone. In the last two groups, only incidental tumors of the esophagus were observed. Nasal cavity tumors also appeared earlier in the animals treated with DSF and NDEA than in animals treated with NDEA alone or with NDEA plus PA. At a biochemical level, DSF led to a significant inhibition of hepatic anilinehydroxylase and nitroso-dimethylaminedemethylase in contrast to PA, which had no influence on these enzymes. The reduced activities of these drug-metabolizing enzymes did not appear to be related to gross cytochrome P450 content. Highly significant increases in glutathione content and glutathione-S-transferase activity (GSH/GST) were induced by DSF but not by PA. Because N-nitrosodiethylamine requires enzymatic activation to form the ultimate carcinogen, it is suggested that the observed inhibition of nitrosamine-transforming enzymes in the liver during DSF treatment leads to an increased amount of intact nitrosamines in other organs, e.g., in the esophagus, where it could be transformed to the ultimate carcinogen. DSF treatment alone or in combination with NDEA leads to an accumulation of trace elements in the liver, whereas PA eliminated copper and cobalt. The possible influence of these elements on tumor development is discussed in part II of this study.

Effect of disulfiram on the alkylation of rat liver DNA by nitrosodiethylamine

ZusammenfassungDie Alkylierungsrate von Guanin der Rattenleber DNA in 7 N-und O-6-Stellung wurde untersucht nach chronischer Gabe von unmarkiertem Nitrosodiäthylamin und einer abschließenden Dosis 14C markierter Substanz. Disulfiram vermindert die Alkylierungsrate in der DNA der Rattenleber. Es konnte ferner gezeigt werden, daß eine nicht fortlaufende Verabreichung von Disulfiram nur eine bestimmte Zeitdauer vor der Alkylierung durch 14C-Nitrosodiäthylamin schützt.SummaryThe extent of ethylation of guanine in 7 N and O 6 position in rat liver DNA was studied after chronic feeding with unlabelled nitrosodiethylamine followed by a single application of the 14C labelled drug. Disulfiram inhibits the alkylation of rat liver DNA. It was also shown that a discontinuous dosage of disulfiram protects against the alkylating effect of 14C nitrosodiethylamine only for a distinct period of time.

Inhibition of Chemical Carcinogenesis

The inhibition of chemical carcinogenesis by specific antidotes has to be considered a long-term objective in cancer prevention. Taking three examples, the possibilites and limitations of such inhibition are elucidated.

Metabolism of debrisoquine and susceptibility to breast cancer

SummaryThere may exist an association between the genetically determined oxidation status of the antihypertensive agent debrisoquine (DEB) and the propensity to develop tumours. The metabolism of DEB is extensive in 90% of healthy subjects (metabolic ratio=MR=0–12.6; MR=% DEB excreted divided by % 4-hydroxy-DEB excreted) and poor in 10% (MR >12.6). In patients with cancer of the lung, urinary bladder, and gastrointestinum, the percentage of high metabolizers is increased to >98%. The poor metabolizer mode is almost devoid of cancer patients. It was investigated whether breast cancer patients show a similar association with respect to the oxidative status of DEB. 108 breast cancer patients and 123 women with benign gynecologic disorders received 1 tablet of 10 mg DEB orally in the evening. Urine was collected for the subsequent 8 hrs and analysed for its content of DEB and its main urinary metabolite 4-OH-DEB by means of HPLC. No decreased amount of poor metabolizers was seen in the cancer group.

Use of diethyldithiocarbamate as a probe to detect stable intermediates during the decomposition of several mutagenic and nonmutagenic N-nitroso compounds

SummaryBy showing that methyldiethyldithiocarbamate is formed from the reaction of methylnitrosourea and disulfiram, we demonstrated in previous experiments that one of the anticarcinogenic/antimutagenic mechanisms of disulfiram is the scavenging of reactive species. We propose that this reaction may be employed additionally as a model for elucidating the following: (a) possible reactions between alkylating species and nucleophilic sites within the cell, and (b) the existence of stable intermediates during the metabolism of N-nitroso compounds. With structurally related pairs of nitrosoureas (n-propyl/isopropyl; cyclopropyl/allyl; 2-phenylethyl/1-phenylethyl), for which each alkylating group of the first compound can spontaneously rearrange to form the alkylating group of the second isomer, we investigated whether the alkylation proceeds via a monomolecular (sn1) or a bimolecular substitution (sn2). For this, we comparatively determined the relative mutagenic activities of each isomer in Salmonella typhimurium TA 1535, as well as their reactivities towards diethyldithiocarbamate (DDTC) by identifying the reaction products. These studies were aimed at revealing the possible formation of a free carbonium ion in the decomposition of several nitrosoureas in the rat liver supernatant fraction. Our system showed that DDTC reacts by two competing mechanisms: attack at the diazonium ion and at the free carbonium ion.Therefore the striking differences which were observed in the mutagenic potency of cyclopropylnitrosourea and N-nitrosoallylurea as well as of N-nitroso-2-phenylethylurea and N-nitroso-1-phenylethylurea cannot be explained only by the different electrophilic reactivities of the respective intermediates.

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.