Vitamin D3 protects against nitrogen mustard-induced apoptosis of the bronchial epithelial cells via activating the VDR/Nrf2/Sirt3 pathway.

Respiratory system injury is the main cause of mortality for nitrogen mustard (NM)-induced damage. Previous studies indicate that reactive oxygen species (ROS) participates in NM-mediated respiratory injuries, but the detailed mechanism is not quite clear. Human bronchial epithelial cell lines 16HBE and BEAS-2B were treated with HN2, a type of NM. In detail, it was shown that HN2 treatment induced impaired cell viability, excessive mitochondrial ROS production and enhanced cellular apoptosis in bronchial epithelial cells. Moreover, impaired Sirt3/SOD2 axis was observed upon HN2 treatment, with decreased Sirt3 and increased acetylated SOD2 expression levels. Sirt3 overexpression partially ameliorated HN2-induced cell injury. Meanwhile, vitamin D3 treatment partially attenuated HN2-induced apoptosis and improved the mitochondrial functions upon HN2 intervention. In addition, HN2 exposure decreased VDR expression, thus inhibiting the Nrf2 phosphorylation and Sirt3 activation. Inhibition of Nrf2 or Sirt3 could decrease the protective effects of vitamin D3 and enhance mitochondrial ROS production via modulating mitochondrial redox balance. In conclusion, impaired VDR/Nrf2/Sirt3 axis contributed to NM-induced apoptosis, while vitamin D3 supplementation provides protective effects via the activation of VDR and the improvement of mitochondrial functions. This study provides novel mechanism and strategy for NM exposure-induced pulmonary injuries.

Preclinical pharmacokinetic and toxicology assessment of red blood cells prepared with S-303 pathogen inactivation treatment.

BACKGROUND: A system has been developed to inactivate a wide spectrum of blood-borne pathogens in red blood cells (RBCs) before transfusion. The system utilizes S-303 to target nucleic acids of pathogens and white blood cells. The safety of pathogen inactivated RBC was assessed using S-303-treated RBCs (S-303 RBCs) and S-300, the primary degradation product of S-303. STUDY DESIGN AND METHODS: As part of a preclinical safety evaluation program, intravenous toxicity, safety pharmacology, toxicokinetic, and pharmacokinetic studies were conducted in rats and dogs with S-303 RBCs and S-300. RESULTS: Single and repeated transfusions of S-303 RBCs were well tolerated in rats and dogs at S-303 concentrations up to five times higher than that used to prepare RBCs for clinical use. For S-300, the doses ranged from the lowest level representative of a clinical exposure from transfusion of 1 unit (0.052 mg/kg/day) to up to the amount of S-300 that would result from exposure to more than 1900 units of RBCs (100 mg/kg/day). There were no related effects of S-303 RBCs or S-300 on mortality, clinical status, body weight, or clinical laboratory assessments and no evidence of organ toxicity. S-300 did not accumulate in the plasma of rats and dogs after repeated transfusions. For all the studies, plasma S-303 was consistently below the limit of quantitation. CONCLUSION: The level of residual S-303 and S-300 in the treated blood component is well below that at which no adverse effects were observed. These results support further clinical development of S-303 RBCs for prevention of transfusion-transmitted infections.

Identification and quantitation of the N-acetyl-L-cysteine S-conjugates of bendamustine and its sulfoxides in human bile after administration of bendamustine hydrochloride.

We recently reported the detection of mercapturic acid pathway metabolites of bendamustine, namely, cysteine S-conjugates in human bile, which are supposed to subsequently undergo further metabolism. In this study, we describe the identification and quantitation of consecutive bendamustine metabolites occurring in human bile using authentic reference standards and the synthesis and structural confirmation of these compounds. Mass spectrometry data along with high-performance liquid chromatography retention data (fluorescence detection) of the synthetic reference standards were consistent with those of the metabolites found in human bile after administration of bendamustine hydrochloride to cancer patients. Analysis of the purified synthetic reference compounds showed a purity of at least 95%. Structural confirmation was achieved by one- and two-dimensional proton as well as carbon-13 NMR spectroscopy and mass spectrometry. A total of 16 bendamustine-related compounds were detected in the bile of patients, 11 of them were recovered as conjugates. Eight conjugates have been structurally confirmed as novel mercapturic acids and sulfoxides. Biliary excretion of the sulfoxides was twice that of the mercapturate precursors. Glutathione S-conjugates of bendamustine have not been detected in bile samples, indicating rapid enzymatic cleavage in humans. Both the lack of glutathione (GSH) conjugates and occurrence of diastereomeric sulfoxides emphasize species-related differences in the GSH conjugation of bendamustine between humans and rats. The total amount recovered in the bile as the sum of all conjugates over the period of 24 h after dosing averaged 5.2% of the administered dose. The question of whether the novel metabolites contribute to urinary excretion should be a target of future investigations.

Free radical production from the interaction of 2-chloroethyl vesicants (mustard gas) with pyridine nucleotide-driven flavoprotein electron transport systems.

The biochemical sequelae to chloroethyl mustard exposure correspond very well to toxic processes initiated by free radicals. Additionally, mustard solutions contain spontaneously formed cyclic onium ions which produce carbon free radicals when reduced electrochemically. Therefore, we hypothesized that the onium ions of sulfur or nitrogen mustards might produce carbon free radicals upon being reduced enzymatically, and that these radicals might constitute a metabolic activation. We set out to document radical production using an in vitro metabolic system and electron paramagnetic resonance (EPR). Our system consisted of NADPH, one of several pyridine nucleotide-driven flavoprotein reductases, cytochrome c as a terminal electron acceptor, various sulfur or nitrogen mustards and the spin trap alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone in buffer. Reactions were started by adding the reductase to the other materials, vortexing and immediately transferring the mixture to a 10 mm EPR flat cell. Repeated scans on a Bruker ESP 300E EPR spectrometer produced a triplet of doublets with hyperfine splitting constants of a(N)=15.483 G and a(H)=2.512 G. The outcome supported our hypothesis that carbon-centered free radicals are produced when mustard-related onium ions are enzymatically reduced. The EPR results varied little with the chloroethyl compound used or with porcine or human cytochrome P450 reductase, the reductase domain of rat brain neuronal nitric oxide synthase or rat liver thioredoxin reductase. Our results offer new insight into the basis for mustard-induced vesication and the outcome of exposure to different mustards. The free radical model provides an explanation for similarities in the lesions arising from mustard exposure and energy-based lesions such as those from heat, ultraviolet and nuclear radiation as well as damage across tissue types such as skin, eyes or airway epithelium.

Mass spectrometry studies of the binding of the minor groove-directed alkylating agent alkamin to AT-tract oligonucleotides.

Minor groove binding alkylating agents, which have potential as cancer drugs, generate cytotoxic DNA adducts that are relatively resistant to repair as a consequence of locating covalent attachment at purine N3 nitrogen atoms. Recently, we used electrospray and matrix-assisted laser desorption ionization mass spectrometry to study the binding of the minor groove-directed polybenzamide bis-half-mustard alkamin, and its monofunctional analogue alkamini, to the oligonucleotide d(CGCGAATTCGCG)(2), identifying a number of inter- and intrastrand alkamin cross-links involving the GAATTC sequence [ Abdul Majid , A. M. S. , Smythe , G. , Denny , W. A. , and Wakelin , L. P. G. ( 2007 ) Mol. Pharmacol. 71 , 1165 - 1178 ]. Here, we extend these studies to d(CGCAAATTTGCG)(2), A3T3, and d(CGCAAAAAAGCG).d(CGCTTTTTTGCG), A6/T6, in which the opportunity for both inter- and intrastrand cross-linking is enhanced. We find that both ligands alkylate all adenines in the longer AT-tracts, as well as the abutting guanines, whether they are in the same strand as the adenines or not, in a manner consistent with covalent attack on purine N3 atoms from the minor groove. Alkamin forms intrastrand cross-links involving A4 and A6 and A6 and G10 in A3T3 and all of the purines in the A6/T6 purine tract, including G10. In addition, it forms interstrand cross-links between A4, A5, A6 and A4', A5', A6', between G10 and the latter adenines in A3T3, and between G22 and adenines A5 and A6 in A6/T6. The reactivity of the abutting guanines provides unexpected opportunities for both inter- and intrastrand cross-linking by alkamin, such as the interstrand cross-link in the CAAAAAAG sequence. We conclude that positioning monofunctional mustard groups on either end of a minor groove-directed polybenzamide has the capacity to enhance interstrand cross-links at all manner of AT-tracts, including most in which the adenines are all in one strand.

[Study on the influence factors of human peripheral blood B lymphocyte transformation by Epstein-Barr virus].

OBJECTIVE: To investigate the influence factors of human peripheral blood B lymphocyte transformation by epstein-barr virus (EBV). METHODS: EB virus was obtained from B95-8 supernatant and quantitated by quantitative PCR amplification. The concentration of cyclosporine A (CSA) concentration of lymphocytes and different time of adding CSA to culture media were selected to examine the effects on the lymphocyte transformation, and the condition of lymphocyte transformation of worker exposed to trichloroethylene was further confirmed. RESULTS: The titer of two virus samples were 5.45 x 10(6) and 1.06 x 10(6) copies/ml. Peripheral blood mononuclear cells (PBMCs) was transformed by using CSA at different concentration and adding time to culture fluid. The concentration of PBMCs being transformed was 2 x 10(6)/ml for normal people, but 1 x 10(6)/ml for worker exposed to trichloroethylene. Cell transformation was confirmed by the gradual increase of cell size and cellular clusters. CONCLUSION: The results suggest the key issue is PBMCs being transformed at a concentration of 2 x 10(6)/ml, rather than using CSA at different concentration and adding time to culture fluid. The PBMCs at concentration of 1 x 10(6)/ml for worker exposed to trichloroethylene were transformed, which may be associated with sensitized status of lymphocytes.

Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors.

BACKGROUND: Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis. RESULTS: To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice. CONCLUSION: Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.

Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin.

We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.

Isolation and characterization of novel phenotype CHO cell mutants defective in peroxisome assembly, using ICR191 as a potent mutagenic agent.

To elucidate molecular and cellular mechanisms of peroxisome biogenesis, we have isolated Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by making use of enhanced green fluorescent protein (EGFP) and a frameshift-inducing mutagen ICR191. CHO-TKa cells stably expressing Pex2p were transformed with a cDNA encoding EGFP fused with peroxisomal targeting signal type 2 (PTS2-EGFP), termed Tka/EG2. TKa/EG2 cells were mutagenized with ICR191 and cultured in the presence of P9OH (9-(1'-pyrene) nonanol) followed by an exposure to UV. P9OH/UV-resistant and morphologically peroxisome-deficient mutant cells were isolated by directly observing cytosolic localization of EGFP, without cell staining. By a combination of cell-fusion and PEX transfection, we determined complementation groups (CGs) of 16 cell mutants isolated here. The mutants were classified into five CGs, including pex2, pex3, pex5, pex6, and pex7 cell mutants. In contrast to typical pex6 mutants with the impaired import of both PTS1- and PTS2-proteins, two clones, ZPEG236 and ZPEG244, showed a distinct, novel phenotype where PTS1-protein import was normal despite the abrogated PTS2 import. Dysfunction of Pex3p in pex3 ZPEG 238 was due to one base (G) insertion in the codon for Asn7 resulting in a frameshift, thereby inducing a distinct 31 amino-acid sequence and a termination. pex2 ZPEG239 showed a mutation in codon GAG for Glu(201) to a nonsense mutation, TAG. Thus, the method developed here using ICR191 could be useful for isolation of further novel cell mutants impaired in peroxisome biogenesis.

Phase II trial of second-line bendamustine chemotherapy in relapsed small cell lung cancer patients.

PURPOSE: The efficacy and toxicity of bendamustine chemotherapy in relapsed small cell lung cancer (SCLC) was determined in this phase II trial. PATIENTS AND METHODS: Patients with cytologically or histologically proven SCLC, who had a sensitive relapse, which was defined as a relapse>or=2 months after completion of primary therapy, were eligible for this study. After informed consent patients received 120 mg/m2 of bendamustine on Days 1 and 2 every 3 weeks. A maximum of six cycles was administered. Primary endpoint was response rate, secondary endpoints included toxicity, progression free survival and overall survival (OS). RESULTS: Twenty-one patients with a median age of 59 years (range 47-76) were accrued to this trial. Six (29%) of 21 patients achieved a confirmed partial remission, 6 (29%) had stable disease and 9 (42%) patients progressed according to RECIST criteria. Median progression free survival was 4 months (95% CI 0-8, 3), median overall survival was 7 months (95% CI 5, 8-8, 2). One- and 2-year survival was 16% and 8%, respectively. Grade III/IV neutropenia occurred in 3 (15%) of 21 patients, 1 patient had a lethal Gram-negative sepsis in neutropenia. Two additional patients had pneumonia in the absence of neutropenia. Two patients (10%) had a grade III anemia, no grade III or IV thrombocytopenia was observed. CONCLUSION: This trial demonstrates efficacy of bendamustine in relapsed SCLC and a favourable toxicity profile. Therefore, single-agent bendamustine is a treatment option for patients with SCLC, who have responded to initial platinum containing chemotherapy and should further be investigated in randomized trials.

DNA adducts formed by a novel antitumor agent 11beta-dichloro in vitro and in vivo.

The multifunctional molecule 11beta-dichloro consists of a ligand for the androgen receptor linked to a bifunctional alkylating group, permitting it to create DNA adducts that bind the androgen receptor. We propose that binding of the androgen receptor to 11beta-DNA adducts acts to both shield damaged sites from repair and disrupt the expression of genes essential for growth and survival. We investigated the formation 11beta-DNA adducts in tumor xenograft and nontumor tissues in mice. Using [14C]-11beta-dichloro, we show that the molecule remains intact in blood and is widely distributed in mouse tissues after i.p. injection. Covalent 11beta-guanine adducts identified in DNA that had been allowed to react with 11beta-dichloro in vitro were also found in DNA isolated from cells in culture treated with 11beta-dichloro as well as in DNA isolated from liver and tumor tissues of mice treated with the compound. We used accelerator mass spectrometry to determine the levels of [14C]-11beta-DNA adducts in LNCaP cells treated in culture as well as in liver tissue and LNCaP xenograft tumors in treated mice. The level of DNA adducts in tumor tissue was found to be similar to that found in LNCaP cells in culture treated with 2.5 micromol/L 11beta-dichloro. Our results indicate that 11beta-dichloro has sufficient stability to enter the circulation, penetrate tissues, and form DNA adducts that are capable of binding the androgen receptor in target tissues in vivo. These data suggest the involvement of our novel mechanisms in the antitumor effects of 11beta-dichloro.

Mass Spectrometry-Based Tools to Characterize DNA-Protein Cross-Linking by Bis-Electrophiles.

DNA-protein cross-links (DPCs) are unusually bulky DNA adducts that form in cells as a result of exposure to endogenous and exogenous agents including reactive oxygen species, ultraviolet light, ionizing radiation, environmental agents (e.g. transition metals, formaldehyde, 1,2-dibromoethane, 1,3-butadiene) and common chemotherapeutic agents. Covalent DPCs are cytotoxic and mutagenic due to their ability to interfere with faithful DNA replication and to prevent accurate gene expression. Key to our understanding of the biological significance of DPC formation is identifying the proteins most susceptible to forming these unusually bulky and complex lesions and quantifying the extent of DNA-protein cross-linking in cells and tissues. Recent advances in bottom-up mass spectrometry-based proteomics have allowed for an unbiased assessment of the whole protein DPC adductome after in vitro and in vivo exposures to cross-linking agents. This MiniReview summarizes current and emerging methods for DPC isolation and analysis by mass spectrometry-based proteomics. We also highlight several examples of successful applications of these novel methodologies to studies of DPC lesions induced by bis-electrophiles such as formaldehyde, 1,2,3,4-diepoxybutane, nitrogen mustards and cisplatin.

Modulation by taurine of the toxicity of taumustine, a compound with antitumor activity.

The antitumor activity of 2-[bis-(2-chloroethyl)-amino]ethanesulfonic acid (also referred to here as "taurine mustard" or "taumustine") was evaluated in the murine P388 and L1210 lymphocytic leukemias and in the pigmented and nonpigmented B16 melanoma systems. Treatment with a single ip dose of taumustine (40 mg/kg) resulted in a 130% increase in life-span for mice bearing P388 (intraperitoneal), a 93% increase for mice bearing L1210 (intraperitoneal), and an approximately 80% increase for mice bearing B16 melanoma (intraperitoneal). Repeated low doses (10 mg/kg) of taumustine promoted a 250% increase in life-span for mice bearing P388 (intraperitoneal), the absence of ascitic fluid from day 4 onward, and the presence of pulmonary emboli from day 5 onward. The inclusion of taurine (5 mM) in the culture medium of P388 cells in primary culture for 45 hours did not alter the cytotoxicity of taumustine, and pretreatment of the tumor-bearing host with taurine (250 mg/kg) in daily treatments with taumustine for up to 8 days did not interfere with antitumor activity (140-160% increased life-span). However, treatment of tumor-bearing mice with taurine abrogated neurotoxicity, intestinal necrosis, pulmonary emboli formation, and tail vein necrosis due to the administration of taumustine. The modulation by taurine of taumustine activity suggests that the combination of these agents offers an advantage of selectivity and host protection during chemotherapy.

Experimental antitumor activity against murine tumor model systems of 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4(3 H)-one (mitozolomide), a novel broad-spectrum agent.

8-Carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H) -one (mitozolomide) demonstrates curative action against a range of murine tumor model systems. At single doses of between 20 and 40 mg/kg, the latter of which approximates the 10% lethal dose value in mice, the compound elicited cures against the L1210 and P388 leukemias irrespective of the route of tumor and/or drug administration; in these tests, animals receiving 10(5) cells i.p. survived greater than 60 days after treatment. Potent effects were also observed against the TLX5 lymphoma (s.c.) and B16 melanoma (i.p.). In other experiments, 7 of 10 animals implanted with 2 X 10(5) Lewis lung carcinoma cells survived greater than 60 days while 10 of 10 animals survived greater than 60 days after implantation of the Colon 26 tumor. Potent inhibition of the solid tumor models was also observed with complete cures of the Colon 38, M5076 sarcoma, and ADJ/PC6A plasmacytoma. In cross-resistance studies, the compound was ineffective against an L1210 leukemia made resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea and against a TLX5 lymphoma resistant to dimethyltriazenes but cured animals bearing the L1210 leukemia with derived resistance to cyclophosphamide.

Antitumor activity of tetraacetylglucosamine mustard.

1,3,4,6-Tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranose is active against L1210 leukemia, giving over 100% increased life-span at optimal dose. Against P388 leukemia, it gives 200% increased life-span with long-term survivors. The compound is most active when given i.p., but shows some activity when given s.c. than p.o., and is more potent (therapeutic and toxic effect) than mechlorethamine on both a molar and a mg basis. Of importance, the schedule dependency for the administration of 1,3,4,6-tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranose in L1210 leukemia differs from most alkylating agents in that it is best given by multiple daily injections rather than as a single large injection on Day 1. This characteristic can be attributed to the amino-glucose moiety.

O6-alkylguanine-DNA alkyltransferase attenuates triazene-induced cytotoxicity and tumor cell immunogenicity in murine L1210 leukemia.

Methylating and chloroethylating triazene compounds (TZCs) are effective antitumor agents in murine leukemias and can induce the appearance of novel antigens in leukemic cells (chemical xenogenization). Recently, it has been shown that TZCs might have a role in the treatment of patients affected by acute myelogenous leukemias that express low levels of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (OGAT). In this report, we have evaluated the role of this DNA repair enzyme in the leukemic cell response to the xenogenizing and cytotoxic properties of TZCs. OGAT-deficient murine leukemic L1210 cells were transfected with a recombinant ecotropic retrovirus containing the coding region for the human OGAT protein. Selected clones expressed the human OGAT transcript and had greatly increased OGAT activity. Compared to OGAT-deficient cells, OGAT-expressing cells were considerably more resistant to the xenogenizing properties of 1-(p-chlorophenyl)-3,3- dimethyl-triazene, measured in terms of leukemia graft rejection, and were less susceptible to the cytotoxic activity of the TZCs 8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one and 8-carbamoyl-3-(2-chloroethyl)imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one. These data suggest that methylation of the O6 position of guanine is involved in the appearance of increased tumor immunogenicity after exposure to methylating TZC and that OGAT is able, at least in part, to counteract the cytotoxic effects of methylating and chloroethylating agents.

Cell cycle-dependent cytotoxicity of alkylating agents: determination of nitrogen mustard-induced DNA cross-links and their repair in Chinese hamster ovary cells synchronized by centrifugal elutriation.

Chinese hamster ovary cells were synchronized into the different phases of the cell cycle by centrifugal elutriation and treated with nitrogen mustard (HN2) in order to investigate the role of DNA damage and repair processes in the cell cycle-dependent cytotoxicity of this alkylating antitumor agent. In agreement with previous studies, cell populations enriched in G1 were the most sensitive to HN2, and those enriched in late S phase-G2 were more resistant, as determined by clonogenic assay. Although the variation in surviving fraction through the cell cycle in response to a single dose (3 micrograms/ml; 1.0 h) of HN2 was as great as a factor of 10, complete dose-response curves generated for the most sensitive and most resistant elutriator fractions indicated that such changes could be accounted for by a ratio of D0 values of only 1.4 Cells synchronized by this same method were also analyzed for their relative levels of HN2-induced DNA cross-linking using the sensitive technique of alkaline elution. There was no significant difference in the levels of either DNA interstrand or DNA-protein cross-links induced in the two elutriator fractions described above immediately after the HN2 treatment. When the amount of DNA cross-linking in the two fractions was measured 6 h after treatment, considerable repair had occurred; however, there was no measurable difference in the rate of repair of either type of cross-link (i.e., DNA interstrand and DNA-protein) in the different phases. Differences in DNA damage and repair processes could not therefore be resolved within the confidence limits of available assays and probably cannot account for the differential cytotoxicity of HN2 towards cells in the different cell cycle phases.

Phase I trial of spiromustine (NSC 172112) and evaluation of toxicity and schedule in a murine model.

Phase I evaluation of spiromustine was performed using an every-3-week schedule and a weekly X 3 schedule. Neurotoxicity was the dose-limiting toxicity presenting as alterations in cortical integrative functions (orientation, language, coordination), leading to a decrease in the level of consciousness. Traditional criteria for grading neurotoxicity poorly characterized these toxicities. The maximum tolerated dose was 6 mg/m2 every 3 weeks and 3 mg/m2 weekly X 3. Concurrent murine studies confirmed spiromustine as a schedule independent drug with toxicity correlating with peak plasma levels. Physostigmine had little effect on decreasing neurotoxicity in the murine model. The solvating agent used was not responsible for the neurotoxicity. Injection of spiromustine on a split-dose schedule decreased the acute neurological toxicity in mice and allowed a larger total dosage to be delivered (compared to single bolus dosage). Based on these results a split-dose schedule is suggested for future clinical trials.

Antitumor activity and bone marrow toxicity of aminoglucose mustard anticancer agents in mice.

In previous structure-activity studies, we have demonstrated that attachment of a glucose molecule to the chloroethylnitrosourea cytotoxic group produces a compound with reduced murine bone marrow toxicity and retention of full antitumor activity. To further define this protective role conferred by the glucose moiety in bone marrow cells, we have replaced the nitrosourea cytotoxic group with another class of alkylating agent, a bifunctional nitrogen mustard. In a detailed structure-activity analysis, we have now characterized four analogues, with the mustard cytotoxic group positioned at carbon 2 [1,3,4,6-tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranos e (TGM)], carbon 6, or carbon 1 (D- and L-isomers) of the aminoglucose molecule. On a molar basis, TGM was most toxic to normal BALB/c X DBA/2 F1 mice, with a 10% lethal dose (LD10) of 3.8 mumol/kg. The D- and L-isomers of 2,3,4,6-tetra-O-acetyl-N,N-bis(2-chloroethyl)glucopyranosylamine (C-1) were the least toxic, with an LD10 of 73 mumol/kg for both. Optimal antitumor activity against the murine P388 leukemia (single i.p. administration of the LD10) did not differ significantly among the four analogues, with increased life span ranging from 83-86%. P388 antitumor activity for nitrogen mustard (HN2) was significantly less, 60% increased life span (P = 0.01), while p-di(2-chloroethyl)amino-L-phenylalanine produced an increased life span of greater than 101%. An LD10 of 6-bis-(2-chloroethyl) amino-6-deoxy-D-glucose (C-6) or TGM produced significantly less depression of WBC counts than did an equitoxic dose of the C-1 isomers, HN2, or p-di(2-chloroethyl)amino-L-phenylalanine. The mean nadir WBC count for C-6 equaled 86% of control, and for TGM, 80% of control. Consistent with this sparing effect on the peripheral WBC, C-6 and TGM produced significantly less in vivo murine bone marrow DNA synthesis depression, 77 and 64% of control, respectively, as compared to the depression nadir produced by HN2 (27% of control), the D-isomer of C-1 (17%), the L-isomer of C-1 (18%), and p-di(2-chloroethyl)amino-L-phenylalanine (2%). These structure-activity studies demonstrate that conjugation of the mustard cytotoxic group to carbon 6 or carbon 2 of glucose produces an analogue that retains P388 antitumor activity significantly greater than that of HN2, with a concomitant reduction in murine bone marrow toxicity.

Inflammatory mechanisms of pulmonary injury induced by mustards.

Exposure of humans and animals to vesicants, including sulfur mustard (SM) and nitrogen mustard (NM), causes severe and debilitating damage to the respiratory tract. Both acute and long term pathological consequences are observed in the lung following a single exposure to these vesicants. Evidence from our laboratories and others suggest that macrophages and the inflammatory mediators they release play an important role in mustard-induced lung injury. In this paper, the pathogenic effects of SM and NM on the lung are reviewed, along with the potential role of inflammatory macrophages and mediators they release in mustard-induced pulmonary toxicity.