Nor-nitrogen Mustard Research Articles

Anthracyclines React with Apurinic/Apyrimidinic Sites in DNA.

The combination of doxorubicin (Adriamycin) and cyclophosphamide, referred to as AC chemotherapy, is commonly used for the clinical treatment of breast and other cancers. Both agents target DNA with cyclophosphamide causing alkylation damage and doxorubicin stabilizing the topoisomerase II-DNA complex. We hypothesize a new mechanism of action whereby both agents work in concert. DNA alkylating agents, such as nitrogen mustards, increase the number of apurinic/apyrimidinic (AP) sites through deglycosylation of labile alkylated bases. Herein, we demonstrate that anthracyclines with aldehyde-reactive primary and secondary amines form covalent Schiff base adducts with AP sites in a 12-mer DNA duplex, calf thymus DNA, and MDA-MB-231 human breast cancer cells treated with nor-nitrogen mustard and the anthracycline mitoxantrone. The anthracycline-AP site conjugates are characterized and quantified by mass spectrometry after NaB(CN)H3 or NaBH4 reduction of the Schiff base. If stable, the anthracycline-AP site conjugates represent bulky adducts that may block DNA replication and contribute to the cytotoxic mechanism of therapies involving combinations of anthracyclines and DNA alkylating agents.

Environmental degradation of human metabolites of cyclophosphamide leads to toxic and non-biodegradable transformation products

The present study assessed the ready biodegradability of the prodrug cyclophosphamide (CPA) and its stable human metabolites in the closed bottle test (CBT). The results of the CBT showed that only the main human metabolite, carboxyphosphamide (CXP), was biodegradable to a certain extent (23 ± 2.4 % ThODNH3). All other metabolites showed neither biodegradation under these conditions nor were any toxic effects on the inoculum observed. Yet, HRMSn results revealed partial primary elimination of all human metabolites and formation of 25 new transformation products. Abiotic degradation via SNi and SN2 reactions was proposed as the main degradation pathway during the CBT. The main degradation products were assigned as 3-(2-chloroethyl)oxazolidin-2-one (COAZ), cytotoxic N-2-chloroethylaziridine (CEZ) and nor‑nitrogen mustard (NNM), an analogue of the chemical warfare agent HN2. While the acute ecotoxicity of the detected products is widely unknown, many have already been reported in medical literature to be either mutagenic, genotoxic, cytotoxic or carcinogenic and may therefore cause a greater risk than their precursors. QSAR models predicted that 16 of them are mutagenic and genotoxic, thus classifying the majority of the chemicals as potential environmental hazards. The central intermediates during the degradation process were proposed as CEZ and its corresponding aziridinium ion. However, other degradation products may occur depending on the type and strength of nucleophiles present in the matrices. Overall, the results demonstrated the importance to include human metabolites in the evaluation of the environmental fate of pharmaceuticals and their risk assessment especially when investigating prodrugs. The results underline the importance of identifying possible degradation products of metabolites, as they can be more toxic than related parent compounds and metabolites and can cause a greater risk to the environment and humans.

Covalent DNA-Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells.

N,N-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active metabolites of cyclophosphamide, a DNA alkylating drug commonly used to treat lymphomas, breast cancer, certain brain cancers, and autoimmune diseases. PM and NOR are reactive bis-electrophiles capable of cross-linking cellular biomolecules to form covalent DNA-DNA and DNA-protein cross-links (DPCs). In the present work, a mass spectrometry-based proteomics approach was employed to characterize PM- and NOR-mediated DNA-protein cross-linking in human cells. Following treatment of human fibrosarcoma cells (HT1080) with cytotoxic concentrations of PM, over 130 proteins were found to be covalently trapped to DNA, including those involved in transcriptional regulation, RNA splicing/processing, chromatin organization, and protein transport. HPLC-ESI(+)-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N7 position of guanines in DNA. Cys-NOR-N7G adduct numbers were higher in NER-deficient xeroderma pigmentosum cells (XPA) as compared with repair proficient cells. Furthermore, both XPA and FANCD2 deficient cells were sensitized to PM treatment as compared to that of wild type cells, suggesting that Fanconi anemia and nucleotide excision repair pathways are involved in the removal of cyclophosphamide-induced DNA damage.

Phosphoramide Mustard As a Biomarker Of Cyclophosphamide Exposure In Adults Receiving Reduced Intensity Conditioning Hematopoietic Cell Transplantation

IntroductionThe use of RIC reduced intensity conditioning (RIC) hematopoietic cell transplant (HCT) has extended the availability of transplantation to older and less medically fit patients. Despite the increased tolerability of RIC HCT, treatment related mortality (TRM) still remains a significant problem. Biomarkers of chemotherapy exposure which are predictive of toxicity or efficacy would be clinically useful in making treatment decisions. We studied phosphoramide mustard (PM), one of the final active metabolites in the complex metabolic pathway of cyclophosphamide (CY), as a biomarker of toxicity and/or efficacy. PM is highly active and undergoes further non-enzymatic degradation to produce nornitrogen mustard which alkylates the guanine on DNA, thereby preventing DNA replication and ultimately leading to cellular death. This is the first report of PM in RIC HCT recipients. MethodsWe studied 10 patients undergoing allogeneic RIC HCT at the University of Minnesota from March 2013 to June 2013 for PM pharmacokinetics. Median age was 60 years (range 34 - 72). Four patients were male and six female. Patients were transplanted for MDS (n=3), AML (n=2), CLL, CMML, B-cell ALL, Hodgkins lymphoma and mantle cell lymphoma. Allogeneic donor sources were double umbilical cord blood (n=4), sibling peripheral blood (n=4) and matched unrelated donor peripheral blood (n=2). RIC preparative regimen consisted of CY 50 mg/kg x1 dose infused over 2 hours on day-6. CY doses were based on actual body weight except for 2 obese patients whose doses were based on an adjusted body weight and received 15 and 25% dose reductions. Preparative regimen also included fludarabine 30-40 mg/m2/day x5 on days -6 to -2 plus single fraction TBI 200 cGy. PM plasma pharmacokinetics were conducted with the single CY dose at times 0 (prior to infusion), 4, 6, 8 and 26 hours after start of CY infusion. PM was derivatised with DDTC and measured by HPLC with ultraviolet detection. Pharmacokinetic measures were calculated using noncompartmental analysis. ResultsMedian (range) total body weight was 76.7 kg (47.9-108.7) and CY dose was 3680mg (2395 - 5210mg). PM was readily measurable in all plasma samples. AUC0-inf was 108.6 hr*mcg/mL (54.0 – 133.9), Cmax was 6.3 mcg/mL (3.7-7.5), concentration at 24 hours was 0.87 mcg/mL (0.06-2.0) and half-life 7.5 hr (2.9 - 12.9). Since not all patients received the same dose, AUC0-inf was normalized to total CY dose and was 0.028 hr*mcg/mL/mg (0.019 - 0.037). There was a weak correlation between dose normalized AUC0-inf, and CrCl, SCr, BMI and BSA (all r2<0.17) but a good inverse correlation with total protein at time of transplant (r2=0.63). There was a good correlation between PM concentration at 26 hours and AUC0-inf (r² = 0.73).Seven patients are alive. Two patients died with TRM at days 37 and 55 posttransplant. Causes of death were graft failure, septic shock secondary to multiple infectious organisms (HHV6, VRE, mucor), and progressive neurological decline, HHV6 encephalopathy and recurrent PRES, respectively. A third patient died of disease progression at posttransplant day 39. The two patients with TRM had 2 of the 3 highest AUCs; 133.9 and 129.8 hr*mcg/mL, respectively. ConclusionWe have demonstrated that PM is readily measurable for at least 26 hours in the plasma after CY treatment. Preliminary analysis suggests that PM exposure may be associated with pretransplant total protein. Renal function appears to play a small role in PM exposure although the range of renal function in this cohort was limited. Additional data are needed to determine the relationship between PM exposure and clinical outcomes.Special acknowledgment for financial support from the American Society of Hematology and the Trainee Research Award program (AW). Disclosures:No relevant conflicts of interest to declare.

Ablation of the ATP-binding cassette transporter, Abca2 modifies response to estrogen-based therapies

The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein expressed in oligodendrocytes and Schwann cells, prostate, ovary and macrophages. In cell cultures, ABCA2 over-expression has been linked with resistance to the anticancer agent, estramustine phosphate (EMP; a nor-nitrogen mustard conjugate of estradiol). The present study shows that Abca2 knockout (KO) mice have greater sensitivity to a variety of side effects induced by EMP treatment. Chronic EMP (12×100mg/kg body weight) produced mortality in 36% of KO mice, but only 7% of age-matched wild type (WT). Side effects of the drug were also more prevalent in the KO mouse. For example, during the first week of EMP treatments, 67% of KO males (compared to 6% of WT males) responded with episodic erectile events. In WT mice, ABCA2 protein localized within pene corpuscles, (which rely on modified Schwann cells for amplification of tactile signals) suggesting that the transporter may function in the erectile process. Endothelial nitric oxide synthase (eNOS; a source of nitric oxide during erectile response) levels were similar in WT and KO male penile tissue. Treatment with 100mg/kg EMP (once daily for four days) elevated serum estradiol and estrone in both WT and KO. However, the circulating levels of these estrogens were higher in KO mice implying a reduced plasma clearance of estrogens as a consequence of ABCA2 ablation. Consistent with the pro-convulsant effects of estrogens, KO mice also displayed an increased incidence of seizures following EMP (14% vs. 0%). Taken together, these data indicate that ABCA2 deficiency renders mice more sensitive to EMP treatment-induced effects implying that the transporter has a role in regulating EMP transport and/or metabolism.

Quantitative High-Performance Liquid Chromatography−Electrospray Ionization Tandem Mass Spectrometry Analysis of Bis-N7-Guanine DNA−DNA Cross-Links in White Blood Cells of Cancer Patients Receiving Cyclophosphamide Therapy

Cyclophosphamide (CPA) is a DNA alkylating agent widely used in cancer chemotherapy. CPA undergoes metabolic activation to phosphoramide mustard and nornitrogen mustard (NOR) which alkylate the N-7 position of guanine in DNA to produce N-[2-(N7-guaninyl) ethyl]-N-[2-hydroxyethyl]-amine (G-NOR-OH) monoadducts and N,N-bis[2-(N7-guaninyl) ethyl] amine cross-links (G-NOR-G). G-NOR-G cross-links are strongly cytotoxic and are thought to be responsible for the biological activity of CPA. In the present work, an isotope dilution high-performance liquid chromatography-electrospray ionization (positive ion) tandem mass spectrometry (HPLC-ESI(+)-MS/MS) methodology was developed to accurately quantify G-NOR-G adducts in human blood. In our approach, DNA extracted from white blood cells (5-20 microg) is spiked with an internal standard of [(15)N(10)]-G-NOR-G and subjected to thermal hydrolysis to release G-NOR-G adducts from the DNA backbone. Following solid phase extraction, G-NOR-G conjugates are quantified by capillary HPLC-ESI-MS/MS in the selected reaction monitoring mode. The application of the new methodology is demonstrated for DNA extracted from blood of three cancer patients receiving 50-60 mg/kg of intravenous CPA. The highest numbers of G-NOR-G adduct (up to 18 adducts per 10(6) normal nucleotides) were observed 4-8 h following CPA administration, followed by a gradual decrease over time, probably due to adduct hydrolysis, DNA repair, and white blood cell death. This methodology will be useful for future investigations of the interindividual differences for CPA-induced DNA-DNA cross-linking.

Synthesis and cytotoxic activity of a glucuronylated prodrug of nornitrogen mustard

A new glucuronylated prodrug of nornitrogen mustard, incorporating the same spacer group as the doxorubicin prodrug HMR 1826, has been prepared. Upon exposure to E. coli β-glucuronidase, fast hydrolysis occurs but a lower cytotoxicity against LoVo cancer cells is observed compared to the nornitrogen mustard alone. This is explained by cyclization of the intermediate carbamic acid to the inactive chloroethyl oxazolidinone.

Estramustine-binding protein in meningioma.

The presence of estramustine-binding protein has been suggested to positively correlate with the effect of the cytotoxic drug estramustine, a combination of estradiol and nornitrogen mustard used in the treatment of prostatic carcinoma. This study demonstrates expression of estramustine-binding protein in a series of meningioma using different ligand-based and immunological techniques. Scatchard plot analysis showed specific binding sites for [3H]lestramustine in meningioma tissue with a dissociation constant of 22-26 nM. Immunohistochemistry revealed an immunoreactivity in meningioma comparable to that demonstrated in prostatic carcinoma. The mean concentration (n = 6) of estramustine-binding protein in meningioma, as determined by radioimmunoassay was 159 ng/g tissue (range 18-274 ng/g). Moreover, partial characterization using size exclusion chromatography of [3H]estramustine-labeled tumor extracts and Western blot analysis of immunoprecipitated samples indicated that the structure of the estramustine-binding protein in meningioma is similar to that in rat prostate, with three polypeptide components of 10, 14, and 16 kDa, as compared to 8, 10-11, and 12 kDa in rat prostate. In conclusion, the novel observation of estramustine-binding protein and specific binding of estramustine in meningioma justify further evaluation regarding the role of estramustine-binding protein in the growth behavior of meningioma and the potential for estramustine and similar hormone-related drugs in the treatment of relapsing meningioma.

Distribution of estramustine in the BT4C rat glioma model.

Estramustine (EaM), a carbamate ester of 17beta-estradiol and nor-nitrogen mustard, is a cytotoxic compound with antitumoral effect in malignant glioma in vitro and in vivo . However, knowledge of the pharmacokinetics of EaM in experimental glioma is limited. The objective of this study was therefore to investigate further the distribution of EaM in the BT4C rat glioma model. Assessment of EaM uptake and distribution was performed by quantitative whole-body autoradiography. In addition, the uptake of EaM and its metabolites estromustine (EoM), estradiol, and estrone were analyzed by gas chromatography. EaM was taken up from the circulation and was found to be the main product in glioma tissue. Whole-body autoradiography after [14C]-EaM administration revealed a strong 14C label simultaneously in tumor and normal brain tissue at 0.5 h after drug administration. In tumor tissue, sustained high levels of 14C label were detected at 12 h after drug administration. In contrast to the tumor, radioactivity in normal brain tissue rapidly leveled off, indicating a retention of radioactivity in the tumor. The tumor/brain radioactivity ratio reached a peak of 4.5 at 12 h after drug administration. High levels of 14C label were also found in pulmonary tissue. By gas chromatography, EoM was found to be the main metabolite in plasma. However, EaM reached higher levels in tumor tissue, with the mean tumor/plasma ratio being 11.7 as compared with 2.0 for EoM. Only low plasma levels of the estrogen metabolites were detected. In conclusion, EaM is taken up in the BT4C rat glioma tissue and is retained in the tumor as compared with normal brain tissue and plasma. EaM showed a greater selectivity for tumor tissue, exhibiting a high tumor/plasma ratio as compared with EoM. The distribution pattern after administration of EaM, as evaluated by both whole-body autoradiography and gas chromatography, supports the earlier suggestion that the uptake is related to a protein with EaM-binding characteristics.

The partitioning of phosphoramide mustard and its aziridinium ions among alkylation and P-N bond hydrolysis reactions.

NMR (1H and 31P) and HPLC techniques were used to study the partitioning of phosphoramide mustard (PM) and its aziridinium ions among alkylation and P-N bond hydrolysis reactions as a function of the concentration and strength of added nucleophiles at 37 degrees C and pH 7.4. With water as the nucleophile, bisalkylation accounted for only 10-13% of the product distribution given by PM. The remainder of the products resulted from P-N bond hydrolysis reactions. With 50 mM thiosulfate or 55-110 mM glutathione (GSH), bisalkylation by a strong nucleophile increased to 55-76%. The rest of the PM was lost to either HOH alkylation or P-N bond hydrolysis reactions. Strong experimental and theoretical evidence was obtained to support the hypothesis that the P-N bond scission observed at neutral pH does not occur in the parent PM to produce nornitrogen mustard; rather it is an aziridinium ion derived from PM which undergoes P-N bond hydrolysis to give chloroethylaziridine. In every buffer studied (bis-Tris, lutidine, triethanolamine, and Tris), the decomposition of PM (with and without GSH) gave rise to 31P NMR signals which could not be attributed to products of HOH or GSH alkylation or P-N bond hydrolysis. The intensities of these unidentified signals were dependent on the concentration of buffer.

Pharmacokinetics and pharmacodynamics of estramustine phosphate.

Estramustine phosphate (estramustine phosphate sodium), a carbamate ester combining 17 beta-estradiol and nor-nitrogen mustard, is a cytotoxic drug used in the treatment of advanced prostatic carcinoma. Because of the radiosensitising effect of this drug there has been a recent increase in interest concerning estramustine phosphate and its clinical use. It has also been found that the early recommendations of drug administration together with food or milk were inappropriate, since calcium containing food and antacids hamper drug uptake. This may have obscured results from earlier clinical studies with estramustine phosphate. Estramustine phosphate is currently being re-evaluated for the treatment of other tumours such as glioma and mammary carcinoma. This review summarises the present relatively limited knowledge concerning the pharmacokinetic and pharmacodynamic aspects of estramustine phosphate and its metabolites.

Tumor blood flow and the cytotoxic effects of estramustine and its constituents in a rat glioma model.

Estramustine (EaM) is a conjugate of nor-nitrogen mustard (NNM) and 17 beta-estradiol (E2) that has cytotoxic and radiosensitizing effects on experimental malignant glioma. Its mechanism of action is only partly understood. To further investigate the mechanism in vivo, the effects on tumor blood flow (TBF) and tumor growth were analyzed. TBF was measured by radioactive microspheres, and tumor growth was measured by weight. Apoptosis was evaluated by in situ end labeling and gel electrophoresis. The effects of the constituents NNM and E2 were also evaluated. EaM increased TBF to 153.8 ml/100 g/min after 3 days and to 153.9 ml/100 g/min after 10 days of treatment, compared with 94.0 ml/100 g/min in untreated controls. Cerebral blood flow did not change after EaM treatment. NNM increased TBF but also showed a tendency to increase cerebral blood flow. E2 increased TBF, whereas cerebral blood flow was unchanged. EaM resulted in a rapid reduction in tumor weight from 230 mg in untreated animals to 146 mg after 3 days of treatment. EaM induced an early transient fragmentation of deoxyribonucleic acid in glioma but not in the normal brain. Neither NNM nor E2 affected tumor weight. EaM increases TBF in the BT4C rat glioma model with a concomitant rapid antitumoral effect. The increase in TBF could partially be induced by an estrogen-like action of EaM, but the rapid cytotoxic effect of the drug is obviously attributed to the intact EaM compound. This cytotoxic effect might be attributable to the induction of programmed cell death.

Estramustine in malignant glioma

Estramustine, a carbamate ester combining 17 beta-estradiol and nornitrogen mustard, has primarily been employed in the treatment of advanced prostatic carcinoma. However, a significant amount of preclinical investigation has been directed toward estramustine's activity against human malignant glioma. These studies have demonstrated that estramustine has potent antiproliferative effects against malignant glioma both in vitro and in vivo. Similar antimitotic effects also have been demonstrated for other carbamate esters. Estramustine does not impair proliferation of nonneoplastic astrocytes at concentrations that inhibit glioma cells. Although the reasons for this selective activity remain to be determined, it has been shown that malignant gliomas expresses an estramustine-specific binding site, estramustine-binding protein, more than brain tissue. In the clinical situation, an uptake and accumulation of estramustine in human glioma tissue have been demonstrated. Estramustine has been shown to enhance the cytotoxic effects of irradiation in relatively radioresistant glioma cells both in cell culture and in a rat glioma model. Estramustine has been regarded as mainly an anti-mitotic drug but recently other effects such as inhibition of DNA synthesis, induction of apoptosis, and membrane alterations have been shown. This report summarizes the preclinical observations concerning the effects of estramustine and related compounds on human malignant gliomas. These findings form the basis for proposing further laboratory and clinical investigation regarding estramustine and human malignant gliomas.

Influence of Aroclor 1254, phenobarbital, β-naphthoflavone, and ethanol pretreatment on the biotransformation of cyclophosphamide in male and female rats

The aim of the present study is to investigate the influence of the environmental factors, smoking and alcohol, on the biotransformation of cyclophosphamide (CP) in the rat in vivo and in vitro with S9 liver fractions. The biotransformation of CP was studied by the determination of the CP metabolites, nor-nitrogen mustard (NNM), 4-ketocyclophosphamide (KCP) and carboxyphosphamide (CAR). The effect of the environmental factors, smoking and alcohol consumption, on the biotransformation enzymes was mimicked by pretreatment of rats with β-naphthoflavone and ethanol, respectively. Rats treated with olive oil and water served as controls and rats pretreated with Aroclor 1254 and phenobarbital were used as positive controls. The influence of sex and supplementation with NAD and GSH, mimicking a biological variation in NAD and GSH levels in rat and human liver, was also studied. Pretreatment of rats with Aroclor 1254 decreased the excretion of unmetabolized CP in urine, most likely due to an enhanced biotransformation. The in vitro hepatic biotransformation of CP in rats was strongly influenced by sex, by supplementation with NAD and GSH and by pretreatment with the enzyme-inducers, phenobarbital and Aroclor 1254. No influence of pretreatment with the enzyme-inducers, β-naphthoflavone and ethanol, was found. The results suggest that the influence of the environmental factors, alcohol consumption and smoking, on the biotransformation of CP in man will be negligible.

Gas chromatographic-mass spectrometric assay for N-2-chloroethylaziridine, a volatile cytotoxic metabolite of cyclophosphamide, in rat plasma

A sensitive and specific method for the quantitative analysis of N-2-chloroethylaziridine (CEA), a volatile cytotoxic metabolite of cyclophosphamide, has been developed using gas chromatography-mass spectrometry and stable isotope dilution techniques. The high volatility problem of CEA during isolation procedure was overcome by the combined use of a deuterium-labeled analog as the internal standard and a Snyder column-concentrator assembly. The assay was found to be linear from 16.7 to 2667 ng/ml in rat plasma with a routine detection limit of 5 ng/ml. The within-run precision at 33, 333 and 1333 ng/ml ( n=6) was found to be 4.8, 4.9, and 6.1%, respectively. The between-run precision was 6.4% ( n=6). The dichloromethane extraction recoveries at 33, 333, and 1333 ng/ml were found to be 101, 98, and 91%, respectively (all at n=6). However, the overall recovery through extraction and evaporation was only 18.3, 15.2, and 27.7% at 33, 333, and 1333 ng/ml levels, respectively. The analytical method was used to evaluate the generation of CEA from its precursors in sodium phosphate buffer, in cell culture media, and the degradation of CEA in these media. In pH 7.4, 0.067 M sodium phosphate buffer at 37°C, both phosphoramide mustard (PM) and nornitrogen mustard (NNM) were degraded in an apparent first-order fashion with half-lives of 24.8 and 14.5 min, respectively. The generated CEA was rather stable in this buffer and degraded with a half-life of 20 h. It was found that 32% PM and 91% NNM were converted to CEA in pH 7.4, 0.067 M sodium phosphate buffer at 37°C, respectively, and 41% PM was transformed into CEA in RPMI 1640 tissue culture media containing 10% FBS at 37°C. The generated CEA was very stabble in the culture media with a degradation half-life of 265 h.

Oxazolidonylethyl adducts to hemoglobin and DNA following nornitrogen mustard exposure

Formation of adducts to hemoglobin (Hb) and DNA of nornitrogen mustard (NNM) was studied with the aim of developing a method for monitoring exposure to NNM. Adducts to N-terminal valines in Hb were studied by the N-alkyl Edman method using pentafluorophenyl isothiocyanate (PFPITC) as the derivatizing reagent. In preliminary studies five major Hb adducts were shown to be formed in reaction of NNM with red cell hemolysate in vitro. Following treatment with PFPITC three of these were found to be pentafluorophenylthiohydantoins (PFPTHs) of N-alkylated valines and the fourth probably originates from NNM esters in which PFPITC had reacted with the nitrogen of N-chloroethylaminoethyl. A PFPTH was found to originate from N-2-(3-oxazolidonyl)ethylvaline, Val-OZ. Val-OZ is formed in reaction, with ring closure to oxazolidone, of CO 2 with the 2-chloroethylamino group in the primary Nvaline- N adduct. Besides a few other adducts, Val-OZ was also observed in mouse Hb following injection of NNM, and also after injection of cyclophosphamide. Following reaction in vitro of NNM with DNA, three major adducts to guanine- N-7 were observed; one of them, 7-[ N′-(2-chloroethyl)-2-aminoethyl]-guanine (NNMCl), was converted by carbonate to 7-[2-(3-oxazolidonyl)ethyl]guanine (Gua-OZ). In mice treated with NNM, Gua-OZ was the only DNA adduct observed. Val-OZ is a chemically stable Hb adduct, potentially useful for monitoring exposures to NNM and cyclophosphamide.

Determination of cyclophosphamide metabolites by gas chromatography and thermionic specific detection interindividual differences in hepatic biotransformation of cyclophosphamide in man in vitro

Sensitive methods for the determination of the cyclophosphamide metabolites nornitrogen mustard, 4-ketocyclophosphamide and carboxyphosphamide are presented. After liquid-liquid extraction and derivatization, the metabolites are determined by gas chromatography and thermionic specific detection. The methods were used to study the in vitro biotransformation of cyclophosphamide with S-9 liver fractions of human donors. The results show large interindividual differences in the formation of nornitrogen mustard and carboxyphosphamide. 4-Ketocyclophosphamide was not detected.

Production of cyclophosphamide metabolites by primary hepatocyte cultures from male and pregnant rats: Effect of Aroclor 1254 pretreatment

In a previous paper (VanAerts et al., Toxicology in Vitro 1993, 7, 769–775) we have shown that the embryotoxicity of Cyclophosphamide was greatly enhanced when Cyclophosphamide had been added to a primary hepatocyte culture derived from Aroclor 1254-pretreated male rats (M A) and the medium from this culture was added to a post-implantation rat embryo culture. However, when medium from hepatocytes that had been derived from untreated male rats (M C), untreated pregnant rats (P C) or Aroclor 1254-pretreated pregnant rats (P A) was used embryotoxicity was low. We now present data on the concentrations of 4-ketocyclophosphamide, carboxyphosphamide and nornitrogen mustard in the primary hepatocyte culture media. 4-Ketocyclophosphamide was absent in media from P C or P A and present in low concentrations in media from M C and M A. Carboxyphosphamide and nornitrogen mustard were present in all media, but their concentrations were several times higher in medium from M A than in media from other sources. This indicates that Aroclor 1254 pretreatment enhances Cyclophosphamide metabolism much more in male rats than in pregnant rats. It is suggested that both a greater inducibility of CYP2B1 and augmented stabilization of 4-hydroxycyclophosphamide by glutathione conjugation may be processes that contribute to the observed differences in metabolite concentrations.

Occupational exposure to nor-nitrogen mustard: chemical and biological monitoring.

Nor-nitrogen mustard is an alkylating agent used in kilogram quantities in the production of various cytostatics. It has been shown to be mutagenic in vitro, and therefore must be regarded as a health hazard. We have studied the environment and blood of a small group of individuals working with nor-nitrogen mustard. New chemical methods for measuring surface contamination were used to make comparisons with ambient air levels of nor-nitrogen mustard. Surprisingly high levels of surface contamination were found even after decontamination, which has led to new decontamination routines and the establishment of wipe test limits of 0.5 microgram nor-nitrogen mustard/dm2. The wipe test has proved effective in following the day-to-day routine handling of nor-nitrogen mustard. Blood samples were taken before, during, and after production runs and analyzed for the frequency of chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and somatic (HPRT) mutations. No significant differences in chromosome aberrations, SCEs, or somatic mutations were found in the peripheral blood from production workers before, during, or after handling of nor-nitrogen mustard. Nor was any difference found between the production workers and a local control group. However, the local controls' chromosome and HPRT frequencies appeared to be somewhat high, demonstrating the problems involved in these types of studies, in which the group sizes are small.

Estramustine revisited.

Estramustine phosphate (Emcyt, Estracyt, estradiol-3-bis (2 chloroethyl) carbamate-17-dihydrogen monophosphate), synthesized in the 1960s [1], was designed and developed for the treatment of breast cancer following the observation of the presence of estrogen receptors in normal breast tissue and tumors [2]. The design of the drug was based on the hypothesis that conjugation of 17-B estradiol with the alkylating agent nor-nitrogen mustard would allow the transport of the cytotoxic agent directly to the steroiddependent tumor cells by virtue of binding to estrogen receptors. This mechanism would then result in improved specificity for the target cells, and at the same time, reduce bone marrow and liver toxicity [3]. The concept leading to synthesis of estramustine phosphate was also based on the beneficial effect observed in prostate cancer of a polyestrodiol phosphate (Estradurin) [4].KeywordsProstate CancerMetastatic Prostate CancerCyproterone AcetateNitrogen MustardLHRH AgonistThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.