Decomposition of BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) in aqueous solution

BCNU, labelled with ¹⁴C in the chloroethyl groups, decomposes in neutral aqueous solution to release half of its radioactivity as volatile products. These have been identified by a combination of gas chromatography and mass spectrometry as vinyl chloride, acetaldehyde, dichloroethane, and chloroethanol. This set of products is consistent with the existence of chloroethylcarbonium ions as reactive intermediates which would produce the previously described substituted nucleotides.     

Reaction of BCNU (1,3-bis (2-chloroethyl)-1-nitrosourea) with polycytidylic acid. Substitution of the cytosine ring

BCNU has been reacted with polycytidylic acid and two derivatives of CMP, 3-hydroxyethyl-CMP and 3,N⁴-ethano-CMP, have been identified in the acid hydrolysate of the polymer. Their formation accounts for some of the reaction of BCNU with nucleic acids, and may be related to the mechanism of action of this compound.     

Reaction of 1,3-bis(2-chloroethyl)-1-nitrosourea with synthetic polynucleotides.

The antitumor agent BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) was incubated with poly(C) and poly(G) in aqueous solution at 37 degrees and pH 7 to produce approximately 0.33 and 0.07% substitution, respectively. Under the same conditions, there was relatively little reaction with poly(A) and poly(U). Poylnucleotides reacted with [14C]BCNU were digested by chemical and enzymatic methods, and the derivative nucleotides were isolated by column chromatography. There were identified by a combination of ultraviolet and mass spectroscopy as 3-(beta-hydroxyethyl)CMP, 3,N4-ethano-CMP, and 7-(beta-hydroxy-ethyl)GMP. This would indicate that BCNU generates active two carbon fragments, probably chloroethyl carbonium ions, which are free to react with nucleotides. The production of these substituted bases may be important to the mechanism of action of the therapeutic nitrosoureas since they would probably alter the function of any nucleic acid which contained them.     

Reaction of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) with guanosine: evidence for a new mechanism of DNA modification

When guanosine reacts with 1,3-bis(2-chloro-2,2-dideuteroethyl)-1-nitrosourea in a mixture of pH 7.1 buffer and DMSO, the 7-chloroethylguanosine which is isolated contains two deuterium atoms located next to the guanine ring and beta to the chlorine atom as shown by electron impact mass spectrometry. It is proposed that initial attack by DNA bases occurs on the number 2 carbon of the haloethylnitrosourea with displacement of the chloride ion. In accordance with this proposed mechanism, 7-bromoethylguanosine is isolated as a major product when BCNU is reacted with guanosine in the presence of high concentrations of KBr. These results suggest that the antitumor activity of various haloethylating antitumor agents may be determined by structural changes which affect their mechanisms of reaction with DNA.     

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU)/interleukin-2 chemoimmunotherapy of murine L1210 leukemia

Summary We have developed a chemoimmunotherapy regimen for the treatment of L1210-cell-induced ascites tumors in mice using a combination of sub-toxic doses of interleukin-2 (IL-2) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU is administered intraperitoneally 4 days after tumor implantation and followed 2 days later by single doses of human recombinant IL-2 for 3 consecutive days. An optimum survival of 84% was achieved using 1500 U IL-2. Reduced survival was observed when lower or higher IL-2 dosages were used. No therapy resulted when heat-inactivated IL-2 was used or when IL-2 was used without chemotherapy. Surviving animals were resistant to L1210 leukemia but not P815 mastocytoma tumor challenge suggesting the combined BCNU/IL-2 therapy stimulated tumor-specific immunity.     

Cytotoxic effects of 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU) on cultured human glioblastomas.

Two cultured human glioblastoma cell lines were exposed to graded doses of BCNU, and the sequential cytoxic and cytocidal changes were studied histologically. Changes included cessation of mitotic activity, reduction in population, rounding and clumping of the cells, nuclear pyknosis, and cell death. The intensity of the changes was proportional to the dosage. With 3 daily doses of 18 mug/ml, the population in Tumor A-172 was only 20% of that in the controls, and 75% of the cells present appeared conviable. This dosage is about four times that used in a single course in treating humans. After 3 doses at 60 mug/ml, cell death appeared complete, but transfer without additional treatment brought out 2-min foci of viable cells, with several mitoses. A more anaplastic glioblastoma, T98, was much less sensitive to BCNU, responding only by lowered population density, decreased mitoses and slight-to-moderate cytotoxicity in one experiment.     

Potentiation in the intact rat of the hepatotoxicity of acetaminophen by 1,3-bis(2-chloroethyl)-1-nitrosourea

Studies of the killing of cultured hepatocytes by acetaminophen indicate that the cells are injured by an oxidative stress that accompanies the metabolism of the toxin (J. L. Farber et al. (1988) Arch. Biochem. Biophys. 267, 640-650). The present report documents that the essential features of the killing of cultured hepatocytes by acetaminophen are reproduced in the intact animal. Male rats had no evidence of liver necrosis 24 h after administration of up to 1000 mg/kg of acetaminophen. Induction of mixed function oxidase activity by 3-methylcholanthrene increased the hepatotoxicity of acetaminophen. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) potentiated the hepatotoxicity of acetaminophen in male rats induced with 3-methylcholanthrene. Whereas the pretreatment with BCNU reduced the GSH content by 40%, a comparable depletion of GSH by diethylmaleate did not potentiate the toxicity of acetaminophen. The antioxidant diphenylphenylenediamine (25 mg/kg) and the ferric iron chelator deferoxamine (1000 mg/kg) prevented the liver necrosis produced by 500 mg/kg acetaminophen in rats pretreated with BCNU. Neither protective agent prevented the fall in GSH produced by acetaminophen. It is concluded the conditions of the irreversible injury of cultured hepatocytes by acetaminophen previously reported are not necessarily different from those that obtain in the intact rat with this toxin.     

Genetic effects of some new bifunctional and water-soluble analogs of the anti-cancer agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in Saccharomyces cerevisiae.

A series of 1,1'-polymethylenebis-3-(2-chloroethyl)-3-nitrosoureas, 1-(omega-hydroxyalkyl)-3-(2-chloroethyl)-3-nitrosoureas, 1,1'(4-methyl-m-phenylenebis)-3-(2-chloroethyl)-3-nitrosourea, 2-[3-(2-chloroethyl)]-3-nitrosoureido-2-deoxy-D-glycopyranose (chlorozotocin and 1-(2-methanesulfonyloxyethyl)-3-(2-chloroethyl)-3-nitrosurea were examined for their genetic activities. BCNU was simultaneously tested as an established, clinically used reference compound. A diploid strain of Saccharomyces cerevisiae, heteroallelic at the gene loci ade2 and trp5, was used as a test system for the induction of mitotic gene conversion (intragenic recombination). All compounds showed strong genetic effects. In the series of aliphatic bifunctional nitrosoureas, 1,1'-ethylenebis-3-(2-chloroethyl)-3-nitrosourea (1) was the most active compound. The water-soluble derivatives, including chlorozotocin, displayed all genetic effects of the same order of magnitude. There was no correlation between genetic activity and chemotherapeutic potential of the test compounds.     

T and B lymphocyte populations of tumor-bearing mice treated with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) or pyran

Summary The immunostimulator pyran copolymer and the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) have been studied to determine their effect on T and B lymphocytes from BALB/c mice bearing a syngeneic tumor, Madison lung 109 carcinoma. Indirect immunofluorescent and mitogenic assays revealed that the tumor-bearing controls usually had T and B cell populations lower than those observed for the normal controls. The tumor-bearing group treated with BCNU generally had T and B cell levels lower than the tumor controls. The administration of pyran resulted in an increase of T cells and a temporary increase in the B cell population. Although this increase in the B cell population of tumor-bearing mice treated with pyran is higher than that seen for the untreated tumor controls it was never higher than in the normal control mice. The experimental results indicate that pyran appears to reconstitute the depressed T cell population and has a transitory effect on the B cell population resulting from tumor burden.     

Effect of treatment with dimethyl triazeno imidazole carboxamide (DTIC,NSC-45388) or 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU,NSC-409962) plus vincristine (NSC-67574) on lymphocyte reactivity of melanoma patients

Summary Fourteen patients with surgically incurable malignant melanoma treated with dimethyl triazeno imidazole carboxamide (DTIC) (2 mg/kg/day i.v. × 10) and 18 patients treated with the combination of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) (150 mg/m² i.v.) plus vincristine (2 mg/m² i.v.) on day 1 only, were tested for lymphocyte reactivity against melanoma cells before and after treatment. Neither chemotherapeutic regimen regularly affected lymphocyte reactivity, either in the presence of fetal calf serum or in the presence of the patients' autologous serum when tested 1 month after treatment. Three patients receiving repeated DTIC therapy and six patients receiving repeated BCNU plus vincristine therapy exhibited little variation in lymphocyte reactivity throughout the course of treatment.     

Mutagenic and recombinogenic consequences of DNA-repair inhibition during treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has been used as a model system to explore whether the clinical combination of the antitumour agent BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) with DNA-repair inhibitors would affect the drug's mutagenic or recombinogenic potential. Preliminary experiments suggested that mitotic crossing-over and other mutagenic events are controlled in a separate fashion. BCNU was more toxic in yeast derivatives with specific defects in any of the three recognised major DNA repair pathways than in the DNA-repair-proficient parent strain. However, in a diploid homozygous for rad18, BCNU showed enhanced mutagenic and recombinogenic potential. Both of these effects were reduced in a comparable rad3 strain, and mitotic crossing-over but not other types of mutagenic event eliminated in the rad52 derivative. Experiments were performed in the presence of three DNA-repair inhibitors which are currently in clinical use and which might be available for combination chemotherapy. Hydroxyurea and amsacrine themselves caused mitotic crossing-over and other events, and did not reduce mutagenic or recombinogenic potential of the BCNU. Hydroxyurea actually decreased toxicity of the BCNU. Caffeine, however, showed some effect in enhancing toxicity and decreasing both mutagenic and recombinogenic potential of the drug. Development of more specific repair inhibitors related to amsacrine or to caffeine, using these repair-deficient strains as model systems, might lead to an enhanced clinical potential of this bisalkylating drug and related compounds.     

Relation between the changes in the structure and synthesis of DNA in the cells of mouse leukemia L1210 induced by 1-methyl-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea

The damage of DNA structure and synthesis in murine leukemia L1210 cells upon single administration in therapeutic doses of antitumour agents of N-nitrosourea type, such as 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied. MNU and BCNU were characterized by stronger inhibitory effects on de novo DNA synthesis compared to additional pathway of DNA synthesis in leukemia L1210 cells in vivo. Centrifugation in alkaline sucrose density gradients of L1210 cell lysates has revealed persistent single-strand breaks and alkaline-labile sites in newly replicated DNA. Parental DNA structure was more stable to damaging drug effects than that of newly replicated DNA. The results are consistent with our previous data on the differences in the mechanisms of MNU and BCNU action and the absence of complete cross resistance between the drugs.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.     

Hydroxylation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) by rat liver microsomes

Ring hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur in the presence of liver microsomes prepared from both normal and phenobarbital induced rats. The metabolite was identified by mass spectrometry after selective extraction and purification by liquid chromatography. The microsomal catalyzed reaction was oxygen and NADPH dependent, inhibited by carbon monoxide and induced 4–5 fold by $\text{in vivo}$ phenobarbital pre-treatment. Phenobarbital induced microsomes hydroxylated the substrate at a rate of 17.6 nmoles/min/mg protein at 37°. A Type I difference spectrum was observed with phenobarbital induced microsomes that also displayed a substrate binding constant (K_s of 4 × 10^?5 M.     

Mechanism of action of the nitrosoureas: formation of 1,2-(diguanosin-7-yl) ethane from the reaction of BCNU (1,3-bis-[2-chloroethyl]-1-nitrosourea) with guanosine

A crosslinked dinucleoside, 1,2-(diguanosin-7-yl) ethane, has been isolated from the reaction of guanosine with the antitumor agent, BCNU. The formation of this product suggests that DNA crosslinking, which may be responsible for the cytotoxicity of BCNU, could occur through such dinucleosides.