alpha-Difluoromethylornithine effects on nitrosourea-induced cytotoxicity and crosslinking in a methylation excision repair positive (MER+) human cell line

We investigated the cytotoxic effects of nitrosoureas with and without a 42-hr preincubation with the ornithine decarboxylase (EC 4.1.1.17) inhibitor alpha-difluoromethylornithine (DFMO, 1 mM) in a MER+ (methylation excision repair positive) human cell line. DFMO combined with a chloroethyl nitrosourea [1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-chloroethyl)-1-nitrosourea (CNU)] yielded increased toxicity with D37 ratios of 1.9 and 3.3 respectively. There was no enhanced toxicity with the monofunctional nitrosourea 1-ethyl-1-nitrosourea (ENU). BCNU or CNU did not induce DNA-DNA interstrand crosslinks in cells with or without a DFMO pretreatment. DNA single-strand breakage was not increased by addition of DFMO. BCNU-induced DNA-protein crosslinking was decreased in cells pretreated with DFMO. These findings are similar to those in MER- cells in that the chloroethyl carbonium alkylating species is required for the enhanced cytotoxicity seen with DFMO. The ability to form DNA interstrand crosslinks, however, does not appear to be necessary for this toxicity enhancement.     

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.     

The lethal activity and repair inhibition capacity of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea

Summary The survival response of human colorectal carcinoma cells treated in vitro for 1 h with PCNU was characterized by a threshold exponential curve, Dq = 8 g/ml (1 h) and D ₀ = 22 g/ml (1 h). Continuous treatment induced decreasing degrees of cell kill although PCNU was biologically stable in solution for at least 24 h. Cells treated with PCNU were unable to recover from potentially lethal damage but were quite capable of repairing PCNU-induced sublethal damage. Thus, PCNU with different alkylating and carbamoylating than other nitrosourea congeners had similar cytotoxic and repair inhibition capacities. Any therapeutic gain in the clinical use of PCNU must derive only from its lipophilic properties and not from its superior activity at the cellular level.Abbreviations PCNU 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea (NSC 95466) - CCNU 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (NSC 79037) - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962) cis-acid, 4-(3-(2-chloroethyl)-3-nitrosoureido)-cis-cyclohexane-carboxylic acid (NSC 153174) - MeCCNU 1-(2-chloroethyl)-3-trans-(4-methyl-cyclohexyl)-1-nitrosourea (NSC 95441) - PE plating efficiency - Dq quasithreshold dose equal to the intercept with the abscissa (at 100% survival) of the exponential part of survival curve - D ₀ mean lethal dose equal to the concentration required to reduce survival by 63% on exponential part of survival curve     

Early effect of BCNU on rat astrocytes. Inhibition of S6 kinase activation by growth factors.

In primary cultures of astrocytes, methylmethane, 2-N-methyl 9-hydroxy-ellepticinium acetate, ditercalinium, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) blocked to various extents the activation of S6 kinase by acidic fibroblast growth factor and insulin [or insulin-like growth factor 1 (IGF1)]. The effects of the most active agent, BCNU, were time and concentration dependent. Pretreatment of cells with 50 microM BCNU for 1 hr completely prevented S6 kinase activation by growth factors for at least 2 days. The S6 kinase activity of unstimulated cells was slightly affected. S6 kinase activation by 12-O-tetradecanoylphorbol 13 acetate was also strongly impaired by treating cells with BCNU whereas activation by 8-bromo-cyclic AMP was slightly reduced. Cyclic AMP-dependent protein kinase and phospholipid and Ca(2+)-dependent protein kinase were unaffected. BCNU had no direct effect on IGF1 binding to cell surface receptors or on the S6 kinase activity of cell cytosols.     

The polymorphic human glutathione transferase T1-1, the most efficient glutathione transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea

A member of the Theta class of human glutathione transferases (GST T1-1) was found to display the greatest catalytic activity towards the cytostatic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of the GSTs studied. In this investigation (the most extensive to date), enzymes from four classes of the soluble human GSTs were heterologously expressed, purified, and kinetically characterized. From the 12 enzymes examined, only GST M2-2, GST M3-3 and GST T1-1 had significant activities with BCNU. This establishes that the activity is not a characteristic of a particular class of GSTs. Although GST M3-3 was previously reported to have the greatest activity with BCNU, the current investigation demonstrates that GST M2-2 is equally active and that GST T1-1 has an approximately 20-fold higher specific activity than either of the Mu class enzymes. A more rigorous kinetic analysis of GST T1-1 gave the following parameters with BCNU: a k(cat) of 0.035 +/-0.003s(-1) and a K(M) of 1.0 +/- 0.1mM. The finding that GST T1-1 has the highest activity towards BCNU is significant since GST T1-1 is expressed in the brain, a common target for BCNU treatment. Furthermore, the existence of a GST T1-1 null allele in up to 60% in some populations, may influence both the sensitivity of tumors to chemotherapy and the severity of adverse side-effects in patients treated with this agent.     

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.     

Synthesis and evaluation of 1,2,2-tris(sulfonyl)hydrazines as antineoplastic and trypanocidal agents

Several 1,2,2-tris(sulfonyl)hydrazines, conceived as prodrugs of 1,2-bis(sulfonyl)hydrazines, were synthesized and evaluated for antineoplastic and trypanocidal activities in mice. 1-Methyl-1,2,2-tris(methylsulfonyl)hydrazine emerged as an extremely efficacious antitrypanosomal agent, whereas 1-(2-chloroethyl)-1,2,2-tris(methylsulfonyl)hydrazine was inactive. In contrast, 1-(2-chloroethyl)-1,2,2-tris(methylsulfonyl)hydrazine displayed potent antineoplastic activity, producing several 60-day "cures" of mice bearing leukemia L1210, leukemia P388, or Sarcoma 180. Furthermore, the fact that the tris(sulfonyl) derivatives will not generate isocyanates, which contribute to the host toxicity of nitrosoureas like 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), makes them agents of significant promise in trypanosomal and cancer chemotherapy.     

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.     

Changes in drug sensitivity of a human astrocytoma clone previously treated with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea in vitro

Summary We have shown in earlier studies that repeated weekly exposures of a human astrocytoma clone (AST 3–4) to MeCCNU (10 g/ml for 1 h per week) produced a linear decrease in survival over the first 3 weekly treatments. But, after that time these cells became progressively more resistant to the 10 g/ml concentration of the agent. In the studies reported here we show that these previously treated cells were also less responsive to other doses ranging from 1 to 30 g MeCCNU/ml. This change in sensitivity to MeCCNU was accompanied by collateral changes in response to other agents: resistance to BCNU and Galactitol, increased sensitivity to Adriamycin, and no change to ionizing radiation. These experiments suggest that once repeated treatments with a single agent cause a tumor cell population to become more resistant, sensitivity to other agents may also change unpredictably.Abbreviations DAG Galactitol - Adria Adriamycin - PCNU 1-(2-Chloroethyl)-3-(2,6 dioxo-3-piperidyl-1-nitrosourea) - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea - FMF Flow microfluorometry Dedicated to the memory of Benjamin Drewinko, Physician, Scientist and friend.     

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.     

In vitro cellular characteristics and survival responses of human astrocytoma clones to chloroethyl-nitrosoureas and dianhydrogalactitol

Three permanent clones were derived from a single astrocytoma cell line and were characterized for in vitro cell kinetics, chromosomal properties and for their responses to the anticancer drugs: 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU); 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea) (PCNU); and 1,2:5,6-dianhydrogalactitol (GAL); all of which have been shown to cross the blood brain barrier. The clones showed different population doubling times, saturation densities, plating efficiencies, chromosome counts, ploidy, cell cycle phase distributions and DNA indices. The only positive correlation among these parameters was between the population doubling times and the modal chromosome numbers; the lower the chromosome number, the shorter the doubling time. No correlation was observable between any of the cellular properties and responses to the four drugs. The clones showed a differential sensitivity to the nitrosoureas, seen maximally as a 600-fold difference in survival between two of the clones treated with the same dose of BCNU. In contrast, the clones exhibited almost identical and uniform sensitivities to galactitol, suggesting that this agent exerted its cytotoxic effects by similar mechanisms in each of the clones. By comparison BCNU (at the tested doses and duration of drug exposure used in this study) was found to be the most effective of the agents tested.     

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.     

Glutathione reductase-deficient erythrocytes as host cells of malarial parasites

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] and its less toxic derivative HeCNU [1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea] are clinically-used antitumour drugs. In erythrocytes BCNU is a highly specific inhibitor of the enzyme glutathione reductase [H. Frischer and T. Ahmad, J. Lab. clin. Med. 89, 1080 (1977)]. When treating erythrocytes in vitro, 50% enzyme inhibition was obtained with 1 microM BCNU or 3 microM HeCNU within 2 hr. The two drugs were used for preparing red cell populations with various levels of glutathione reductase activity; complete inhibition (greater than or equal to 98%) was only achieved when the medium contained glucose as a source of reducing equivalents. The erythrocytes were then tested in drug-free media as host cells for the malaria parasite Plasmodium falciparum. In the range of 0-300 mU/ml cells, there was a correlation between glutathione reductase activity and parasite growth; erythrocytes with an activity of less than 20 mU/ml did not serve as host cells for P. falciparum at all although these erythrocytes were viable. When the culture medium was supplemented with 20 mM glutathione (GSH), parasite growth was normal irrespective of the glutathione reductase level in the erythrocytes. This is consistent with the finding that poisoning glutathione reductase led to a 10-fold decrease of the cytosolic GSH level. Our results corroborate the concept that intraerythrocytic inhibition of glutathione reductase mimicks the biochemistry of drug-sensitive glucose-6-phosphate dehydrogenase deficiency (favism), an inherited condition which confers protection from malaria.     

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.     

Metoclopramide as a sensitizer of 1,3-bis(2-chloroethyl)-1-nitrosourea treatment of brain tumors in the rat.

RG2 glioma-like cells grown in in vitro culture can be inoculated into rat brains using stereotactic surgical procedures to produce tumors with a diameter of 12-16 mm2 in 20-21 days. This system has been used to evaluate if metoclopramide (MCA) could sensitize the tumor toxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU alone (15 mg/kg, intravenously), and MCA alone (2 mg/kg, intraperitoneally), and these drug treatments in combination, were administered so that BCNU alone was given as a single dose on day 3 after inoculation of the RG2 cells, MCA alone was given on day 3 at 0 and 3 h followed by five or six treatments per week beginning 24 h after the 3 h dose, and BCNU plus MCA were given according to the combined schedule where the first MCA treatment was scheduled 30 min prior to the BCNU infusion. The design of this study required the drug treated animals to be matched to untreated animals (controls) at the time of inoculation of the RG2 cells. Under these experimental conditions, BCNU alone and MCA alone had no effect on tumor growth, whereas BCNU plus MCA significantly retarded brain tumor growth. The normal tissue toxicity induced by BCNU treatment, evaluated by measurement of body weight and survival, was not potentiated by the combination of BCNU plus MCA. These data extend the previous findings of MCA as a radio- and chemosensitizer to include the sensitization of another cytotoxic agent (BCNU) and of another type of tumor (malignant glioma).     

BCNU-loaded poly(D,L-lactide-co-glycolide) wafer and antitumor activity against XF-498 human CNS tumor cells in vitro

Implantable polymeric device that can release chemotherapeutic agent directly into central nervous system (CNS) has had an impact on malignant glioma therapy. The purpose of our study was to develop an implantable polymeric device, which can release intact 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) for long-term period over 1 month, and to evaluate its cytotoxicity against XF 498 human CNS tumor cells in vitro. BCNU was incorporated into biodegradable poly(D,L-lactide-co-glycolide) (PLGA), by using spray-drying method. BCNU-loaded PLGA microparticles were characterized by scanning electron microscopy (SEM), powder X-ray diffraction, and differential scanning calorimetry. SEM observation of the microparticles showed that the microparticles were spherical, i.e. microspheres. Homogeneous distribution of BCNU in PLGA microsphere was confirmed by significant reduction of crystallinity of BCNU. Microspheres were fabricated into wafers with flat and smooth surface by direct compression method. In vitro release of BCNU in pH 7.4 phosphate buffered saline was prolonged up to 8 weeks after short initial burst period. Antitumor activity of BCNU-loaded PLGA wafer against XF 498 human CNS tumor cells continued over 1 month and, PLGA only did not affect the growth of the cells. Meanwhile, the cytotoxic activity of BCNU powder disappeared within 12 h. These results strongly suggest that the BCNU/PLGA formulations increase release period of carmustine in vivo and also be useful in the development of implantable polymeric device for malignant glioma.     

Glutathione reductase inhibitors as potential antimalarial drugs. Effects of nitrosoureas on Plasmodium falciparum in vitro

Malarial parasites are believed to be more susceptible to oxidative stress than their hosts. BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea) and HeCNU(1-(2-chloroethyl)-3-(2-hydroxythyl)-1-nitrosourea), inhibitors of the antioxidant enzyme glutathione reductase, were found to prevent the growth of Plasmodium falciparum in all intraerythrocytic stages. When exposing infected red blood cells to 38 microM BCNU or 62 microM HeCNU for one life cycle of synchronously growing parasites, the parasitemia decreased by 90%. During the formation of new ring forms, the parasites are even more susceptible to these drugs. The treatment with BCNU or HeCNU produced a rapid depletion of GSH in the parasites and their host cells; in addition, protection against lipid peroxidation was impaired in these cells. Possible mechanisms for the antimalarial action of the inhibitors are discussed. Our results suggest that erythrocyte glutathione reductase, an enzyme of known structure, might be considered as a target for the design of antimalarial drugs.     

Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs Cloretazine and BCNU

The antitumor, DNA-alkylating agent 1,3-bis[2-chloroethyl]-2-nitrosourea (BCNU; Carmustine), which generates 2-chloroethyl isocyanate upon decomposition in situ, inhibits cellular glutathione reductase (GR; EC 1.8.1.7) activity by up to 90% at pharmacological doses. GR is susceptible to attack from exogenous electrophiles, particularly carbamoylation from alkyl isocyanates, rendering the enzyme unable to catalyze the reduction of oxidized glutathione. Evidence implicates inhibition of GR as a cause of the pulmonary toxicity often seen in high-dose BCNU-treated animals and human cancer patients. Herein we demonstrate that the prodrug Cloretazine (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino)carbonyl]hydrazine; VNP40101M), which yields methyl isocyanate and chloroethylating species upon activation, did not produce similar inhibition of cellular GR activity, despite BCNU and Cloretazine being equally potent inhibitors of purified human GR (IC(50) values of 55.5 microM and 54.6 microM, respectively). Human erythrocytes, following exposure to 50 microM BCNU for 1h at 37 degrees C, had an 84% decrease in GR activity, whereas 50 microM Cloretazine caused less than 1% inhibition under the same conditions. Similar results were found using L1210 murine leukemia cells. The disparity between these compounds remained when cells were lysed prior to drug exposure and were partially recapitulated using purified enzyme when 1mM reduced glutathione was included during the drug exposure. The superior antineoplastic potential of Cloretazine compared to BCNU in animal models could be attributed in part to the contribution of the methyl isocyanate, which is synergistic with the co-generated cytotoxic alkylating species, while at the same time unable to significantly inhibit cellular GR.     

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.     

Effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on the levels of glutathione and lipid peroxidation and the activity of glutathione reductase in liver and lung

After subcutaneous injection of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to rats, glutathione reductase activity in lung and liver diminished rapidly. The restoration of enzyme activity occurred more slowly in the lung than in the liver. The pattern for the time-course of total glutathione (GSH) levels was similar between lung and liver, except for a marked depression of hepatic levels 6 h after drug administration. The level of malondialdehyde (MDA) in lung was not affected by BCNU throughout the experimental period (3 days). However, the level in liver had increased significantly by 6 h after drug administration. These observations indicate that lipid peroxidation in lung was not induced by BCNU even when glutathione reductase activity was markedly diminished. In contrast, the lipid peroxidation in liver was induced by BCNU and was preceded by an early marked depression in total GSH.