Evaluation of the role of isocyanates in the action of therapeutic nitrosoureas

The half-lives of chloroethyl and cyclohexyl isocyanate have been determined in tissue culture medium, and the isocyanate concentration produced during the breakdown of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) has been calculated. L1210 or HeLa cells exposed either to the parent nitrosourea or to an equivalent constant isocyanate concentration show no deficiency in the repair of gamma-irradiation damage as measured by DNA strand separation in alkali. Viability studies indicate that the isocyanates play a minor role in the overall cytotoxicity of the nitrosoureas.     

An analysis of 1-(2-chloroethyl)-1-nitrosourea activity at the cellular level

The effect of five different 1-(2-chloroethyl)-1-nitrosoureas on the growth of cultured P388 cells has been analyzed in terms of physical, chemical, and kinetic parameters that are related to the mechanism of action of this class of cancer chemotherapeutic agent. This study correlates structure with activity at the cellular level by using a dose function that is related to the amount of active species, the (2-chloroethyl)diazonium ion, that is formed during the period of exposure of cells to drug rather than to the initial drug dose. 1-(2-Chloroethyl)-1-nitrosourea analogues that rapidly enter the P388 cells are shown to have the same activity relative to the amount of active species formed. When analyzed in this way, activity is not influenced by the structure of the N-3 substituent, lipophilicity, or carbamoylating activity. The agents 1-(2-chloroethyl)-1-nitrosourea (CNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) all produce a 50% cell growth inhibition at 6 to 7 microM active species formed per cell volume. Chlorozotocin required a twofold higher effective dose to produce the same toxic effect. This decreased activity is attributed to the slow uptake of the water-soluble chlorozotocin into P388 and L1210 cells relative to the rate of chlorozotocin conversion to active species in medium. The yields to 2-chloroethanol from CNU, BCNU, and chlorozotocin were shown to be the same, indicating that these agents generate the same yield of alkylating intermediate at 37 degrees C and pH 7.4.     

Mechanism of action of 2-haloethylnitrosoureas on deoxyribonucleic acid. Nature of the chemical reactions with deoxyribonucleic acid.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) covalently cross-links DNA under physiological conditions. Methyl substitution at either carbon atom of the 2-chloroethyl portion of the molecule prevents cross-linking. Haloalkyi nitrosoureas including 3-chloropropyl, 4-chlorobutyl and 5-chloropentyl, although they readily alkylate DNA, exhibit no ability to cross-link DNA. 3-(2-Chloroethyl)-1-methylcytosine hydrochloride and N⁴-(2-chloroethyl)- 1-methylcytosine hydrochloride, similar to intermediates suggested in the cross-linking process, alkylate PM2-CCC-DNA readily. These two cytosine derivatives also cyclize readily to give 3,N⁴-ethano-1-methylcytosine closely similar to a species isolated from the treatment of poly-C with BCNU. A number of processes including the extent of DNA alkylation, measured with [¹⁴C]CCNU labeled in the ethylene portion of the molecule, as well as concomitant DNA single strand scission, and intramolecular alkylation and/or hydrolysis of the chloroethyl cytidine intermediate were investigated as to their effects upon the interstrand cross-linking process.     

Effect of cations on the formation of DNA alkylation products in DNA reacted with 1-(2-Chloroethyl)-1-nitrosourea.

The purpose of this study was to examine the influence of cations on the formation of the individual DNA alkylation products derived from 1-(2-chloroethyl)-1-nitrosourea (CNU). Reaction of calf-thymus DNA with [(3)H]CNU in 10 mM triethanolamine buffer produced 13 DNA adducts. Seven of these adducts were identified as N7-(2-hydroxyethyl)guanine, N7-(2-chloroethyl)guanine, 1, 2-(diguan-7-yl)ethane, N1-(2-hydroxyethyl)-2-deoxyguanosine, 1-(N1-2-deoxyguanosinyl)-2-(N3-2-deoxycytidyl)ethane, O(6)-(2-hydroxyethyl)-2-deoxyguanosine, and phosphotriesters. The ratios of the individual products indicated that the chloroethyl and hydroxyethyl adducts are derived from different alkylating intermediates. The influence of cations on the formation of these DNA alkylation products was investigated by the addition of either NaCl, MgCl(2), or spermine. The results demonstrated that (1) the levels of DNA alkylation were inversely proportional to ionic strength, (2) the extent of inhibition was dependent on the alkylation product, and (3) the order of relative effectiveness of inhibition of DNA alkylation by these cations was as follows: spermine > Mg > Na. These results support a model whereby reactions which proceed via an S(N)2 mechanism are more sensitive to the effects of ionic strength than reactions which proceed via an S(N)1 mechanism. In 9L cells treated with CNU, the same alkylation products were formed as in purified DNA; however, the product distribution was different. We interpret this to indicate that within cells, cations modify the reaction of intermediates derived from CNU with DNA.     

Potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced cytotoxicity in 9L cells by pretreatment with 6-thioguanine

9L Rat brain tumor cells were treated with 0.2 microM 6-thioguanine for 48 hr, which produced a 40% cell kill, a small (15%) inhibition of cell growth, and an accumulation of cells in S-phase. Maximum incorporation of [14C]6-thioguanine into cellular DNA occurred after 24 hr of incubation; 70% of the label was incorporated into DNA as 6-thio-2'-deoxyguanosine. Pretreatment of 9L cells for 48 hr with 0.2 microM 6-thioguanine potentiated the cytotoxicity of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) by 50% with a dose enhancement ratio of 1.5, and caused a 30% increase in the number of BCNU-induced sister chromatid exchanges (SCEs) and a 50% increase in DNA crosslinks formed, compared to treatment with BCNU alone. Used as a single agent, 6-thioguanine induced a significant number of SCEs. Results suggest that these effects may be related to the increased formation of DNA crosslinks, possibly as the result of the formation of S6-(2-chloroethyl)-6-thioguanine in cellular DNA.     

In vitro pharmacokinetics and pharmacodynamics of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

The relationship between treatment efficacy and the pharmacokinetics (PK) and pharmacodynamics (PD) of anticancer drugs is poorly defined. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent used in the treatment of brain and other forms of cancer. It is postulated that BCNU kills cells by forming DNA interstrand cross-links. The present study was undertaken to characterize the PK and PD of BCNU in mouse L1210 cells. L1210 cells were exposed to BCNU (0-160 microM) and analyzed for intracellular BCNU concentrations, DNA interstrand cross-links, cell cycle phase, and cytotoxicity. The half-life of BCNU in cells was approximately 40 min. The maximum reduction of mitochondrial enzyme activity (maximum cell death) achieved within 24 hr after exposure to BCNU was concentration-dependent and could be described by a Hill equation. At lower concentrations, the area under the DNA interstrand cross-link-time curve linearly correlated with the maximum cell death and the area under the BCNU concentration-time curve. BCNU induced cell accumulation in the G(2)/M phase of the cell cycle, which continued even after apparent completion of cross-link repair. Loss of membrane permeability was minimal (approximately 2%) during the first 24 hr. Thereafter, cells died exponentially over the next 9 days, primarily by necrosis. In conclusion, while cytotoxicity was concentration-dependent, an indirect relationship was found among the time-course of BCNU concentrations, DNA interstrand cross-links, and cell death. Because of the disparity between the time-scale of PK and PD, focusing only on the early events may provide limited information about the process of anticancer drug-induced cell death.     

Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(β-d-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L121o leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonucleases micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [¹⁴C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest.64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210: bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210: bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.     

Comparison of DNA lesions produced by tumor-inhibitory 1,2-bis(sulfonyl)hydrazines and chloroethylnitrosoureas

1,2-Bis(sulfonyl)hydrazine derivatives, designed to generate several of the electrophilic species classically believed to be responsible for the alkylating (chloroethylating) and/or carbamoylating activities of the chloroethylnitrosoureas (CNUs), were compared with respect to the cross-linking and nicking of T7 DNA to that caused by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chloroethyl)-3-(4-trans-methylcyclohexyl)-1-nitrosourea (MeCCNU). In the case of BCNU, a large proportion of T7 DNA strand nicking was found to be due to the generation of 2-chloroethylamine, produced from the hydrolysis of 2-chloroethylisocyanate, in turn formed during the decomposition of the parental nitrosourea. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (compound 1) gave a greater yield of DNA cross-links than the CNUs. Compound 1, as well as its derivatives that were incapable of generating 2-chloroethylisocyanate, did not produce detectable levels of strand nicking, indicating that N7-alkylation of guanine did not occur to a significant extent with these agents. Since compound 1 and its derivatives are believed to generate chloronium and chloroethyldiazonium ions, it would appear that these species could not be significantly involved in the N7-alkylation of guanine caused by the CNUs. The relatively low level of N7-alkylation of guanine residues and the relatively high yield of cross-links generated by some of the 1,2-bis(sulfonyl)-1-(2-chloroethyl)hydrazine derivatives implies that they are more exclusive O6-guanine chloroethylating agents than the CNUs. O6-Guanine chloroethylation is believed to be the therapeutically relevant event produced by the CNUs; therefore, compound 1 derivatives represent promising new cancer chemotherapeutic agents, since they appear to generate lower quantities of therapeutically unimportant, yet carcinogenic lesions, and more of the therapeutically relevant O6-guanine chloroethylation than the CNUs.     

Modulation of nitrosourea resistance in human colon cancer by O6-methylguanine.

Human colon cancer is resistant to a variety of alkylating agents including the nitrosoureas. To specifically evaluate nitrosourea resistance, we studied the role of O6-alkylguanine-DNA alkyltransferase (alkyltransferase) which is known to repair nitrosourea-induced cytotoxic DNA damage. Alkyltransferase activity varied over a similar wide range in 25 colon cancer biopsies and 14 colon cancer cell lines but the activity was not correlated with differentiation status, Dukes' classification or in vitro growth characteristics. 1,3-Bis-(2-chloroethyl)-1-nitrosourea (BCNU) resistance and alkyltransferase activity were highly correlated (R2 = 0.929, P less than 0.001) in 7 different colon cancer cell lines, suggesting that the alkyltransferase is an important component of nitrosourea resistance in colon cancer cells. In the BCNU-resistant, high alkyltransferase VACO 6 cell line, inactivation of the alkyltransferase by O6-methylguanine caused a proportional decrease in the BCNU IC50, consistent with that predicted by the regression line. Enzyme inactivation was also associated with a marked increase in DNA cross-link formation. Because alkyltransferase correlates with BCNU resistance in colon cancer, and resistance can be reversed by inactivating the protein, the alkyltransferase may have an important role in nitrosourea resistance in human colon cancer cells. These data provide the rationale for clinical trials in colon cancer with biochemical modulators of the alkyltransferase to increase the therapeutic response to nitrosoureas.     

Studies of the mechanism of action of the tumour-inhibitory nitrosoureas.

BCNU [1,3 bis-(2-chloroethyl)-1-nitrosourea] and some related nitrosoureas have been shown to have a wide spectrum of action against a number of transplanted rodent tumours. No correlation was found between the chemical instability of a nitrosourea and its antitumour activity. Unlike difunctional alkylating agents, the nitrosoureas inhibit the incorporation of tritiated precursors in DNA, RNA and protein to equal extents, the inhibition of tritiated thymidine incorporation into DNA occurring within 5 min of incubating cells with BCNU. Although the biological half life of BCNU was found to be very short (15 min by bioassay) a single injection was as effective against the established and widely-disseminated TLX5 lymphoma as against the early transplant. BCNU interfered specifically with the incorporation of labelled thymidine triphosphate into DNA, but no inhibition of DNA polymerase could be demonstrated at physiological dose levels. In their mechanism of action and in their biological properties the tumour inhibitory nitrosoureas are quite distinct from the bifunctional alkylating agents.     

A comparative study of therapeutic activity,myelotoxicity and DNA damage in the bone marrow of mice after cyclophosphamide and ASTA Z 7557 (INN mafosfamide)

Summary This study compares the two oxazaphosphorine compounds ASTA Z 7557 (AZ) and cyclophosphamide (CP) in their therapeutic activity as well as in their myelotoxicity and DNA damage being induced after a single intraperitoneal injection. Therapeutic activity was determined towards methylnitrosourea-induced rat mammary carcinomas in vivo and in vitro, resulting in comparable efficacy of both compounds at their optimal doses, respectively, with the sensitivity of individual tumors being reflected by the degree of inhibition of ³H-thymidine uptake of these cells in vitro.Myelotoxicity was measured as inhibition of pluripotent (CFU-S) and macrophage-granulocyte committed (CFU-C) stem cells together with the extent of single strand breaks and DNA-DNA interstrand crosslinks in murine bone marrow. At equimolar base AZ was found to induce a higher level of DNA damage than CP in the bone marrow of mice 16 hours after a single intraperitoneal injection. Both compounds depressed the pluripotent stem cell compartment of the bone marrow to a similar extent, whereas AZ was significantly less toxic to the granulocyte cell lineage.Abbreviations AZ ASTA Z 7557; 2-[N,N,bis-(2-chloroethyl)-amino]-4-(2-sulphonato-ethylthio)-tetrahydro-2H-1,3,2, oxazaphosphorine-2-oxide - CP Cyclophosphamide; 2-[N,N,bis-(2-chloroethyl)-amino]-1,3,2-oxazaphosporine-2-oxide - CFU-C Granulocyte-committed stem cells - CFU-S Pluripotent stem cells - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea - MNU N-methyl-N-nitrosourea - Mesna 2-mercaptoethanesulphonate     

Pronounced damage of intestinal tract mucosa by the combination of 1-methyl-1-nitrosourea plus 1,3-bis-(2-chloroethyl)-1-nitrosourea. Effect of drug sequence and time interval of administration

The combination of 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) has an overadditive toxicity in rats. This overadditive effect is dependent on drug sequence and time interval between the administration of both compounds. Application of MNU within 2 h prior to BCNU or simultaneous application of both compounds displayed the highest toxicity. The dose-limiting toxicity appears to be a severe damage of the intestinal mucosa.     

Pathways and kinetics of aqueous decomposition and carbamoylating activity of new anticancer nitroimidazole-linked 2-chloroethylnitrosoureas

The products of decomposition in anaerobic aqueous solution at pH 7.1 and 37 degrees were determined for two series of novel anticancer agents incorporating both nitroimidazole and 2-chloroethylnitrosourea moieties (NI-CENUs) and examples of which exhibit preferential hypoxic toxicity against HeLa-MR cells. The decomposition products identified were vinyl chloride, acetaldehyde, 2-chloroethanol, ethylene glycol and imidazole-bearing compounds of the type including oxazolidinone, ethylamine or urea moieties. Series A NI-CENUs, which contain a 2-hydroxypropyl unit, gave rise to the oxazolidinone intramolecularly compared with the series B agents which gave rise to the imidazole-ethylamine and ureas. The half-lives of the B series agents were comparable with those of 1,3-bis(2-chloroethyl)nitrosourea (BCNU), 2-cyclohexyl-1-(2-chloroethyl)-1-nitrosourea (CCNU) and streptozotocin. The carbamoylation activity of the series B agents was approximately ten times that of series A compounds. This latter property may be related to the greater potency of series B than series A NI-CENUs against Mer+ HeLa-S3 cells via inhibition of relevant repair enzymes.     

Stimulation and inhibition of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced strand breaks and interstrand cross-linking in Col E1 plasmid deoxyribonucleic acid by polyamines and inorganic cations

The influence of various polyamines and metallic cations on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced DNA single-strand breaks and DNA interstrand cross-linking was in Col E1 plasmid using electrophoretic techniques. Spermidine and spermine (0.4 to 1.5 mM concentration range) markedly stimulated BCNU-induced DNA nicking, whereas putrescine had no effect on the nicking process. In contrast to the polyamines, BCNU-induced DNA nicking was decreased by the three inorganic cations, Na+ (100 and 200 mM), Mg2+ (0.5 and 1.5 mM), and Co3+ (NH3)6 (0.2 and 0.4 mM), with the trivalent hexamminecobalt ions being most inhibitory. When the monofunctional N-methyl-N-nitrosourea (MNU) was used (instead of the bifunctionally active BCNU) to alkylate Col E1 DNA, nicking of the DNA was inhibited by spermidine. Furthermore, the ability of chloroethylated Col E1 DNA to form interstrand cross-links after treatment with BCU was inhibited by 0.5 mM spermidine and 0.5 mM spermine, both concentrations within the intracellular range. Putrescine at 3-6 mM only marginally stimulated DNA cross-linking. In comparison, the inorganic cations all enhanced Col E1 DNA cross-linking by BCNU, with the rank order of cross-link stimulation being Mg2+, Na+, and Co3+ (NH3)6. These results provide evidence that polyamines can interact with DNA to modulate chloroethylnitrosourea-induced DNA damage and that the interaction is not only a function of the charge on the polyamine molecule but also of the chemical structure of the polyamine.     

In vitro cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine, towards cells from human oral tissue.

The cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), towards cultured human cells from oral tissue was evaluated. The toxicity of SNAP to Smulow-Glickman gingival epithelial cells was correlated with the liberation of nitric oxide, as N-acetyl-D,L-penicillamine, the SNAP metabolites, N-acetyl-D,L-penicillamine disulfide and nitrite, and preincubated (denitrosylated) SNAP did not affect viability. Comparing equimolar concentrations of various nitric oxide donors, cytotoxicity appeared to be inversely related to the relative stability (i.e., half-life) of the test compound; the sequence of cytotoxicity for a 4 hr exposure was S-nitrosoglutathione>spermine NONOate> SNAP>DPTA NONOate>DETA NONOate. Intracellular reduced glutathione (GSH) was lowered in S-G cells exposed to SNAP. Pretreatment of the cells with the GSH depleter, 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU), enhanced the toxicity of SNAP Similar findings of enhanced sensitivity to SNAP were noted with gingival fibroblasts and periodontal ligament cells pretreated with BCNU. The toxicity of SNAP towards the gingival epithelial cells was decreased by cotreatment with the antioxidants, N-acetyl-L-cysteine, L-ascorbic acid, and (+)-catechin. Cells exposed to SNAP exhibited nuclear aberrations, including multilobed nuclei and multinucleation. SNAP-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis.     

1,3-(2-Chloroethyl)-1-nitrosourea potentiates the toxicity of acetaminophen both in the phenobarbital-induced rat and in hepatocytes cultured from such animals

The toxicity of acetaminophen was studied in hepatocytes cultured from phenobarbital-induced male rats. Such cells were less sensitive to acetaminophen than similar ones cultured from animals induced with 3-methylcholanthrene. In both cases, the toxicity of acetaminophen depended on its metabolism. Inhibition of glutathione reductase with 1,3-(2-chloroethyl)-1-nitrosourea (BCNU) potentiated the toxicity of acetaminophen in the presence or absence of 100 mM acetone, an agent that activates the mixed function oxidation of the toxin. BCNU enhanced the rate and extent of the depletion of GSH in the presence or absence of acetone. Pretreatment of the hepatocytes with the ferric iron chelator deferoxamine or addition to the culture medium of the antioxidant N,N'-diphenyl-p-phenylenediamine prevented the toxicity of acetaminophen in the presence of BCNU whether or not there was acetone in the cultures. BCNU similarly potentiated the hepatotoxicity of acetaminophen in the intact, phenobarbital-induced rat. These data indicate that the mechanism of the killing of hepatocytes induced with phenobarbital is similar to that reported previously with hepatocytes prepared from animals induced with 3-methylcholanthrene. In both cases it would seem that the liver cells are killed by acetaminophen as a result of an oxidative stress that accompanies the metabolism of this hepatotoxin.     

A chemical basis for the antitumor activity of chloroethylnitrosoureas

A comparison of the aqueous decomposition products of several haloethylnitrosoureas has led to a suggested mode of decomposition and antitumor effect for these compounds. 1,3-Bis-chloroethyl-1-nitrosourea (BCNU), 1-chloroethyl-3-cyclohexyl-1-nitrosourea (CCNU), 1,3-bis-fluoroethyl-1-nitro-sourea (BFNU) and 1-chloroethyl-1-nitrosourea (CNU) decompose in buffered aqueous solution to yield haloethanol, vinyl halide, dihaloethane and acetaldehyde. Evidence is presented that these products are derived from an intermediate haloethyl carbonium ion. On the other hand, 1-chloroethyl-3,3-dimethyl-1-nitrosourea decomposes slowly in aqueous solution, generates acetaldeh?yde, but not the other volatile compounds described above, and is not toxic to murine L1210 leukemia cells in vitro. In contrast to the disubstituted nitrosoureas, chloroethylnitrosourea does not generate an organic isocyanate on aqueous decomposition, but is a very active antitumor agent both in vitro and in vitro. These observations support the hypothesis that the antitumor activity of the chloroethylnitrosoureas is due to the facile decomposition of the parent molecule to form a chloroethyl carbonium ion (or diazonium precursor).     

Cytochrome P-450-dependent formation of alkylating metabolites of the 2-chloroethylnitrosoureas MeCCNU and CCNU

Rat liver microsomes catalyzed the biotransformation of the clinically important nitrosourea anticancer agents 1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea (MeCCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) to alkylating metabolites that bound covalently to microsomal protein and to DNA. The enzyme-mediated microsomal alkylation required NADPH and oxygen and was inhibited by carbon monoxide, indicating the participation of a cytochrome P-450-dependent monooxygenase. Additional studies with inhibitors such as piperonyl butoxide and with the inducers 3-methylcholanthrene and phenobarbital were consistent with this view. In contrast to these observations on the formation of alkylating metabolites, carbamylation reactions were not affected significantly by microsomal metabolism. Reduced glutathione, cysteine or N-acetylcysteine decreased the microsomal alkylation by MeCCNU and produced a corresponding increase in the formation of polar metabolites that was resolved by HPLC as three distinct N-acetylcysteine-MeCCNU adducts. The addition of semicarbazide to the reaction decreased microsomal alkylation by 30%, indicating that the formation of the alkylating species may proceed via an aldehyde intermediate. Renal microsomes were not found to catalyze the alkylation reaction. Moreover, MeCCNU inhibited the renal slice accumulation of p-aminohippuric acid only in the presence of liver microsomes and NADPH, suggesting that a liver metabolite may be responsible for the renal toxicity of the parent nitrosourea.     

Carbamoylating chemoresistance induced by cobalt pretreatment in C6 glioma cells: putative roles of hypoxia-inducible factor-1

1. We tested whether pretreatment of reagents known to induce hypoxia-inducible factor-1 (HIF-1) may confer chemoresistance against cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to rat C6 glioma cells. We also studied which cytotoxic mechanism(s) of chloroethylnitrosoureas could be neutralized by cobalt preconditioning. 2. Preconditioning of rat C6 glioma cells with cobalt chloride (300 microm, 2 h) induced HIF-1 binding activity based on electrophoretic mobility shift assay (EMSA). Results from Western blotting confirmed a heightened HIF-1alpha level upon cobalt chloride exposure (300-400 microm, 2 h). Cobalt chloride (300 microm) pretreatment for 2 h substantially neutralized BCNU toxicity, leading to increases in glioma cell survival based on MTT assay. In addition, pre-exposure of C6 cells with desferrioxamine (DFO; 400 microm, 3 h), an iron chelator known to activate HIF-1, also induced HIF-1 binding and rendered the glioma cells resistant to cytotoxicity of BCNU. 3. Pre-incubation with cobalt chloride abolished the cytotoxicity of several carbamoylating agents including 2-chloroethyl isocyanate and cyclohexyl isocyanate, the respective carbamoylating metabolites of BCNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. The protective effect of cobalt exposure, however, was not observed when cells were challenged with alkylating agents including temozolomide. 4. Cadmium chloride (50 microm) effectively reversed cobalt-induced HIF-1 activation. Correspondingly, cadmium chloride suppressed carbamoylating chemoresistance mediated by cobalt chloride pretreatment. Furthermore, both double-stranded oligodeoxynucleotide (ODN) decoy with HIF-1 cognate sequence and antisense phosphorothioate ODNs against HIF-1alpha partially abolished the carbamoylating chemoresistance associated with cobalt preconditioning. 5. Our results suggest that cobalt- or DFO-preconditioning may enhance glioma carbamoylating chemoresistance that is dependent, at least in part, on induction of HIF-1.