Effect of glutathione modulation using buthionine sulfoximine on DNA methylation by dimethylnitrosamine in the rat.

An in vivo study was carried out in order to determine whether glutathione (GSH) might serve as a scavenger for the supposed electrophilic methylating fragment derived from dimethylnitrosamine (DMN) and thus function to decrease the degree of cellular macromolecule interaction, estimated by measuring the DNA methylation yield. After a 4-hr pretreatment with DL-buthionine-SR-sulfoximine (BSO), a specific inhibitor of GSH synthesis, male Sprague-Dawley rats were dosed with radiolabeled DMN (250 micrograms/kg). Four hours later the animals were killed and the livers and kidneys were excised. The DNA isolated from these organs was hydrolyzed in mild acid, and the liberated purines were quantified utilizing HPLC and liquid scintillation counting. The 70-75% GSH depletion in the liver and kidney resulting from BSO pretreatment did not have any significant effect on the degree of DNA methylation as assessed by the 7-methylguanine/guanine yield. In control experiments we found that DMN doses greater than 1 mg/kg had a marked effect on liver and kidney GSH levels after 4 hr.

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

By 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/l-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.

Influence of a prolonged treatment with disulfiram and D(-)penicillamine on nitrosodiethylamine-induced biological and biochemical effects in rats. I. Investigations on the drug metabolizing system.

The influence of a prolonged treatment with disulfiram (DSF) and D(-)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.

[Prevention of tumor formation in the bladder by sodium-2-mercaptoethane sulfonate (mesna). Experimental studies and clinical consequences]. / Prophylaxe der Tumorentstehung in der Harnblase durch Natrium-2-mercaptoethansulfonat (Mesna). Experimentelle Untersuchungen und klinische Konsequenzen.

An experimental model was developed, in which urinary bladder cancer was induced by cyclophosphamide in rats, thus reproducing cyclophosphamide-induced urinary bladder carcinogenesis observed in humans. It was possible in this model to achieve a highly significant reduction of cyclophosphamide-induced urinary bladder cancer by concomitant administration of sodium 2-mercaptoethane sulfonate (mesna). A significant delay of urinary bladder carcinogenesis by administration of the uroprotective substance mesna was also observed when using butylbutanolnitrosamine for inducing urinary bladder cancer. It was thus for the first time possible to assure chemoprevention of this tumor type by administration of a specific antidote.

Chemical interaction of disulfiram with nitrosodimethylamine after in vitro enzymatic activation.

The in vitro reaction between disulfiram (DSF) and N-nitroso[14C]dimethylamine [( 14C]NDMA) was studied. Incubations of DSF with [14C]NDMA were carried out in the presence of rat liver microsomes, control 9000 g (S9) supernatant fraction and phenobarbital-induced S9 fraction. H.p.l.c. analysis and liquid scintillation measurement provided evidence for the formation of methyldiethyldithiocarbamate (MeDDTC) as a product of the reaction between diethyldithiocarbamate (DDTC), the main active metabolite of DSF and the 'methyl-cation' released by NDMA after enzymatic activation. The amount of MeDDTC found here was consistent with the rate of oxidation of NDMA to formaldehyde. Scintillation counting confirmed that other radioactive peaks, not due to MeDDTC, were unrelated to the methylation of L-cysteine by [14C]NDMA.

Delay of bladder cancer induction in rats treated with N-nitroso-N-butyl-N-(4-hydroxybutyl)amine by administration of sodium-2-mercaptoethanesulfonate (Mesna).

The influence of sodium-2-mercaptoethanesulphonate (Mesna) on urinary bladder cancer induced by N-nitroso-N-butyl-N-(4-hydroxybutyl)amine (BBNOH) was studied in male Sprague-Dawley rats. The treatment consisted of 5 g/kg BBNOH per gavage and 63 g/kg Mesna in drinking water over a period of 39 weeks. A positive control group was given the same dose of BBNOH as the treated group. Although Mesna did not reduce the incidence of bladder carcinomas, it significantly increased the lifespan of the animals, thus suggesting a partial general protective action.

In vitro formation of methyldiethyldithiocarbamate after the reaction of nitrosoacetoxymethylmethylamine or methylnitrosourea with disulfiram.

Analysis by scintillation measurement, mass spectrometry and h.p.l.c. showed that diethyldithiocarbamic acid (DDTC), the main metabolite of disulfiram (DSF), forms methyldithiocarbamate (MeDDTC) when incubated with [14C]nitrosoacetoxymethylmethylamine ([14C]NAMM) or [14C]methylnitrosourea ([14C]MNU) in different media (bacteria, esterases, rat liver 9000 x g supernatant fraction and microsomes). When DSF instead of DDTC was used, MeDDTC was formed only when soluble enzymes were present which are required to split DSF into two DDTC moieties. No physiological methylation of DDTC takes place as was shown by experiments with [3H]MNU. [14C]Methanol, formed by the decay of [14C]NAMM and [14C]MNU was shown to have no alkylating properties.