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Molecular and Cellular Biology May 2000Bifunctional alkylating agents and other drugs which produce DNA interstrand cross-links (ICLs) are among the most effective antitumor agents in clinical use. In...
Bifunctional alkylating agents and other drugs which produce DNA interstrand cross-links (ICLs) are among the most effective antitumor agents in clinical use. In contrast to agents which produce bulky adducts on only one strand of the DNA, the cellular mechanisms which act to eliminate DNA ICLs are still poorly understood, although nucleotide excision repair is known to play a crucial role in an early repair step. Using haploid Saccharomyces cerevisiae strains disrupted for genes central to the recombination, nonhomologous end-joining (NHEJ), and mutagenesis pathways, all these activities were found to be involved in the repair of nitrogen mustard (mechlorethamine)- and cisplatin-induced DNA ICLs, but the particular pathway employed is cell cycle dependent. Examination of whole chromosomes from treated cells using contour-clamped homogenous electric field electrophoresis revealed the intermediate in the repair of ICLs in dividing cells, which are mostly in S phase, to be double-strand breaks (DSBs). The origin of these breaks is not clear since they were still efficiently induced in nucleotide excision and base excision repair-deficient, mismatch repair-defective, rad27 and mre11 disruptant strains. In replicating cells, RAD52-dependent recombination and NHEJ both act to repair the DSBs. In contrast, few DSBs were observed in quiescent cells, and recombination therefore seems dispensable for repair. The activity of the Rev3 protein (DNA polymerase zeta) is apparently more important for the processing of intermediates in stationary-phase cells, since rev3 disruptants were more sensitive in this phase than in the exponential growth phase.
Topics: Antineoplastic Agents, Alkylating; Cisplatin; Cross-Linking Reagents; DNA Damage; DNA Repair; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Endodeoxyribonucleases; Exodeoxyribonucleases; Flap Endonucleases; Fungal Proteins; Haploidy; Mechlorethamine; Mutation; Rad52 DNA Repair and Recombination Protein; Recombination, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 10779332
DOI: 10.1128/MCB.20.10.3425-3433.2000 -
Toxicological Sciences : An Official... Nov 2018Nitrogen mustard (NM) is a vesicant known to cause acute pulmonary injury which progresses to fibrosis. Macrophages contribute to both of these pathologies. Surfactant...
Nitrogen mustard (NM) is a vesicant known to cause acute pulmonary injury which progresses to fibrosis. Macrophages contribute to both of these pathologies. Surfactant protein (SP)-D is a pulmonary collectin that suppresses lung macrophage activity. Herein, we analyzed the effects of loss of SP-D on NM-induced macrophage activation and lung toxicity. Wild-type (WT) and SP-D-/- mice were treated intratracheally with PBS or NM (0.08 mg/kg). Bronchoalveolar lavage (BAL) fluid and tissue were collected 14 days later. In WT mice, NM caused an increase in total SP-D levels in BAL; multiple lower molecular weight forms of SP-D were also identified, consistent with lung injury and oxidative stress. Flow cytometric analysis of BAL cells from NM treated WT mice revealed the presence of proinflammatory and anti-inflammatory macrophages. Whereas loss of SP-D had no effect on numbers of these cells, their activation state, as measured by proinflammatory (iNOS, MMP-9), and anti-inflammatory (MR-1, Ym-1) protein expression, was amplified. Loss of SP-D also exacerbated NM-induced oxidative stress and alveolar epithelial injury, as reflected by increases in heme oxygenase-1 expression, and BAL cell and protein content. This was correlated with alterations in pulmonary mechanics. In NM-treated SP-D-/-, but not WT mice, there was evidence of edema, epithelial hypertrophy and hyperplasia, bronchiectasis, and fibrosis, as well as increases in BAL phospholipid content. These data demonstrate that activated lung macrophages play a role in NM-induced lung injury and oxidative stress. Elucidating mechanisms regulating macrophage activity may be important in developing therapeutics to treat mustard-induced lung injury.
Topics: Animals; Bronchoalveolar Lavage Fluid; Female; Lung Injury; Macrophage Activation; Macrophages, Alveolar; Male; Mechlorethamine; Mice; Mice, Inbred C57BL; Oxidative Stress; Pulmonary Surfactant-Associated Protein D
PubMed: 30060251
DOI: 10.1093/toxsci/kfy188 -
Nucleic Acids Research Sep 2011DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen...
DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.
Topics: Antineoplastic Agents; Cisplatin; Cross-Linking Reagents; DNA; DNA Damage; DNA Repair; DNA-Directed DNA Polymerase; Deoxycytosine Nucleotides; Mechlorethamine; Nucleic Acid Conformation; Nucleotidyltransferases; Saccharomyces cerevisiae Proteins; Templates, Genetic
PubMed: 21666254
DOI: 10.1093/nar/gkr448 -
Investigative Ophthalmology & Visual... Apr 2016Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can...
PURPOSE
Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing.
METHODS
Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 μL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays.
RESULTS
Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment.
CONCLUSIONS
Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.
Topics: ADAM Proteins; ADAM17 Protein; Animals; Blotting, Western; Cells, Cultured; Corneal Diseases; Corneal Stroma; Epithelium, Corneal; Humans; Mechlorethamine; Rabbits; Tomography, Optical Coherence; Tumor Necrosis Factor-alpha
PubMed: 27058125
DOI: 10.1167/iovs.15-17269 -
Environmental Health Perspectives Nov 2006Exposure of the brain to environmental agents during critical periods of neuronal development is considered a key factor underlying many neurologic disorders.
BACKGROUND
Exposure of the brain to environmental agents during critical periods of neuronal development is considered a key factor underlying many neurologic disorders.
OBJECTIVES
In this study we examined the influence of genotoxicants on cerebellar function during early development by measuring global gene expression changes.
METHODS
We measured global gene expression in immature cerebellar neurons (i.e., granule cells) after treatment with two distinct alkylating agents, methylazoxymethanol (MAM) and nitrogen mustard (HN2). Granule cell cultures were treated for 24 hr with MAM (10-1,000 microM) or HN2 (0.1-20 microM) and examined for cell viability, DNA damage, and markers of apoptosis.
RESULTS
Neuronal viability was significantly reduced (p < 0.01) at concentrations > 500 microM for MAM and > 1.0 microM for HN2; this correlated with an increase in both DNA damage and markers of apoptosis. Neuronal cultures treated with sublethal concentrations of MAM (100 microM) or HN2 (1.0 microM) were then examined for gene expression using large-scale mouse cDNA microarrays (27,648). Gene expression results revealed that a) global gene expression was predominantly up-regulated by both genotoxicants; b) the number of down-regulated genes was approximately 3-fold greater for HN2 than for MAM; and c) distinct classes of molecules were influenced by MAM (i.e, neuronal differentiation, the stress and immune response, and signal transduction) and HN2 (i.e, protein synthesis and apoptosis).
CONCLUSIONS
These studies demonstrate that individual genotoxicants induce distinct gene expression signatures. Further study of these molecular networks may explain the variable response of the developing brain to different types of environmental genotoxicants.
Topics: Alkylating Agents; Animals; Animals, Newborn; Astrocytes; Cell Survival; Cells, Cultured; Cerebellum; DNA Damage; Gene Expression Profiling; Mechlorethamine; Methylazoxymethanol Acetate; Mice; Mice, Inbred C57BL; Neurons; Oligonucleotide Array Sequence Analysis
PubMed: 17107856
DOI: 10.1289/ehp.9073 -
Nucleic Acids Research Mar 1997Previous work showed that melphalan-induced mutations in the aprt gene of CHO cells are primarily transversions and occur preferentially at G-G-C sequences, which are...
Previous work showed that melphalan-induced mutations in the aprt gene of CHO cells are primarily transversions and occur preferentially at G-G-C sequences, which are potential sites for various bifunctional alkylations involving guanine N-7. To identify the DNA lesion(s) which may be responsible for these mutations, an end-labeled DNA duplex containing a frequent site of melphalan-induced mutation in the aprt gene was treated with melphalan, mechlorethamine or phosphoramide mustard. The sequence specificity and kinetics of formation of both interstrand and intrastrand crosslinks were determined. All mustards selectively formed two base-staggered interstrand crosslinks between the 5'G and the G opposite C in the 5'G-G-C sequence. Secondary alkylation was much slower for melphalan than for the other mustards and the resulting crosslink was more stable. Mechlorethamine and phosphoramide mustard induced intrastrand crosslinks between the two contiguous Gs in the G-G-C sequence in double-stranded DNA, but melphalan did not. Molecular dynamic simulations provided a structural explanation for this difference, in that the monofunctionally bound intermediates of mechlorethamine and phosphoramide mustard assumed thermodynamically stable conformations with the second arm in a position appropriate for intrastrand crosslink formation, while the corresponding melphalan monoadduct did not.
Topics: Adenine Phosphoribosyltransferase; Alkylation; Animals; Base Sequence; CHO Cells; Cricetinae; DNA Damage; DNA Methylation; Kinetics; Mechlorethamine; Melphalan; Models, Molecular; Mutagenesis; Nitrogen Mustard Compounds; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Phosphoramide Mustards
PubMed: 9092631
DOI: 10.1093/nar/25.6.1211 -
DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards is preserved in intact cells.Nucleic Acids Research Jun 1992Nitrogen mustard alkylating agents react with isolated DNA in a sequence selective manner, and the substituent attached to the drug reactive group can impose a distinct...
Nitrogen mustard alkylating agents react with isolated DNA in a sequence selective manner, and the substituent attached to the drug reactive group can impose a distinct sequence preference. It is not clear however to what extent the observed DNA sequence preferences are preserved in intact cells. The highly reiterated sequence of human alpha DNA has been used to determine the sites of guanine-N7 alkylation following treatment of cells with three nitrogen mustards, mechlorethamine, uracil mustard and quinacrine mustard, known to react in isolated DNA with distinctly different sequence preferences. Alpha DNA from drug treated cells was extracted, purified, end-labeled, and a 296 base pair, singly end-labelled, fragment isolated. Following the quantitative conversion of alkylation sites to strand breaks the fragments were separated on DNA sequencing gels. Clear differences were observed between the alkylation patterns of the three compounds, and the selectivities were qualitatively similar to those predicted and observed in the same sequence alkylated in vitro. In particular the unique preferences of uracil and quinacrine mustards for 5'-PyGC-3' and 5'-GT/GPu-3' sequences, respectively, were preserved in intact cells suggesting that the pattern of sequence dependent reactivity is not grossly affected by the nuclear milieu.
Topics: Alkylation; Base Sequence; DNA; Guanine; Humans; Mechlorethamine; Molecular Sequence Data; Quinacrine Mustard; Tumor Cells, Cultured; Uracil Mustard
PubMed: 1620613
DOI: 10.1093/nar/20.12.3175 -
Pharmacological Reports : PR 20092,4,6-Trinitrophenyl (TNP) hapten-labeled peritoneal macrophages (Mf) given intravenously (iv) to recipients are poor inducers of contact sensitivity (CS) reactions...
2,4,6-Trinitrophenyl (TNP) hapten-labeled peritoneal macrophages (Mf) given intravenously (iv) to recipients are poor inducers of contact sensitivity (CS) reactions unless Mf donors are pretreated with low doses of cyclophosphamide (CY). In vivo CY is converted into active alkylating metabolites, phosphoramide mustard (PM) and acrolein (ACR). Our experiments aimed to test how in vitro treatment of non-immunogenic Mf with different concentrations (10(-5) to 10(-7) M) of CY metabolites will influence their immunogenicity and other biological functions. Instead of chemically unstable PM, we used structurally and functionally similar nitrogen mustard (NM). Our experiments show that treatment of Mf with ACR or NM stimulates the in vitro production of pro-inflammatory IL-6 and IL-12 and down-regulates anti-inflammatory IL-10 and TGF-beta cytokines. In vivo non-immunogenic TNP-Mf become capable of inducing CS reactions in two situations: first, after treatment with NM or ACR and second, when cell recipients are received iv before Mf transfer of monoclonal antibodies against IL-10 and/or TGF-beta (500 mug per animal). Treatment with NM, but not with ACR, was also an efficient stimulus for production by Mf of significantly increased levels of reactive oxygen intermediates (ROIs). In summary, our experiments show that CY metabolites can significantly increase the specific immune response as well as nonspecific innate reaction (ROIs production) and support the notion that CY and its metabolites can be a promising accessory tool when upregulation of the immune response is desired.
Topics: Acrolein; Animals; Cells, Cultured; Cyclophosphamide; Dermatitis, Contact; Immunity; Inflammation Mediators; Macrophages, Peritoneal; Male; Mechlorethamine; Mice; Mice, Inbred CBA; Reactive Oxygen Species
PubMed: 19605955
DOI: 10.1016/s1734-1140(09)70098-2 -
Pharmacological Reports : PR 2012According to cytotoxic and mutagenic properties, nitrogranulogen (NTG) changes the character of inflammatory reactions. Our previous studies have shown that NTG can...
BACKGROUND
According to cytotoxic and mutagenic properties, nitrogranulogen (NTG) changes the character of inflammatory reactions. Our previous studies have shown that NTG can enhance immunological defense reactions, because of its high affinity to DNA, and causes disorders in the synthesis of acute phase proteins (e.g., haptoglobin, transferrin, fibrinogen and complement protein C3) [15]. The aim of the current studies was to determine the influence of three different NTG doses: 5 μg/kg b.w. (body weight), 50 μg/kg b.w. and 600 μg/kg b.w. (cytotoxic dose) on the values of hematological blood parameters: RBC, HGB, HCT, RDW, MCV, MCH, MCHC, PLT, MPV, PCT, PDW, WBC, NEUT, LYMPH, MONO, EOS and BASO in pleuritis-induced rats.
METHODS
The animals were randomized into five groups: Group I - control group; Group II - IP (induced pleuritis) group; Group III - NTG5 group; Group IV - NTG50 group; Group V - NTG600 group. The blood was collected from all the groups at the 24(th) h, 48(th) h, and 72(nd) h after the initiation of the carrageenin-induced inflammatory reaction.
RESULTS
These investigations have revealed that NTG administered at the dose of 5 μg/kg b.w. caused the drop of the leukocyte and lymphocyte numbers and the rise of the neutrophil number at the 72(nd) h of the experimental-induced inflammatory reaction. Moreover, the dose of: 5 μg/kg b.w. was an immunomodulatory property and it also increased the erythrocytic parameters. On the contrary, NTG applied at the doses of 50 μg/kg and 600 μg/kg b.w. contributed to the drop of both: the erythrocytic and leukocytic parameters during the whole time of the inflammatory reaction.
CONCLUSIONS
The results suggest that nitrogranulogen affects the erythropoiesis.
Topics: Alkylating Agents; Animals; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Erythrocytes; Erythropoiesis; Female; Inflammation; Leukocytes; Lymphocytes; Mechlorethamine; Neutrophils; Pleurisy; Random Allocation; Rats; Rats, Inbred BUF; Time Factors
PubMed: 22814019
DOI: 10.1016/s1734-1140(12)70861-7 -
Trisethylene-imino-s-triazine (triethylene melamine or TEM) in the treatment of neoplastic diseases.California Medicine Jul 1951Trisethylene-imino-s-triazine (triethylene melamine or TEM) produced minimal effects in inhibiting transplantable lymphoma and mammary adenocarcinoma in mice. In strain...
Trisethylene-imino-s-triazine (triethylene melamine or TEM) produced minimal effects in inhibiting transplantable lymphoma and mammary adenocarcinoma in mice. In strain A mice, injection of the compound induced pulmonary tumors.TEM was tried on 32 patients with neoplastic disease, including nine patients with Hodgkin's disease and five with lymphosarcoma and lymphatic leukemia. The therapeutic and toxic effects were similar to those observed with nitrogen mustard (HN2). Satisfactory remissions of up to three months were observed in Hodgkin's disease and lymphosarcoma following parenteral administration of TEM. It is the authors' impression that the remissions obtained with TEM were not as complete and did not last as long as those obtained with HN2.TEM is effective by the oral route as well as parenterally, and produces much less emetic reaction than HN2. On the other hand, the chemotherapeutic range is narrower than that of HN2. Patients who do not respond to HN2 show no response to TEM.TEM is a drug of some clinical usefulness in the same conditions and with the same general limitations and toxic effects as HN2. The ease of administration of TEM increases its hazards, and close clinical and hematologic observations are essential on patients receiving the agent.
Topics: Animals; Hodgkin Disease; Humans; Lung Neoplasms; Lymphoma; Mechlorethamine; Mice; Neoplasms; Nitrogen Mustard Compounds; Triazines; Triethylenemelamine
PubMed: 14848718
DOI: No ID Found