Breast cancer drugs perturb fundamental vascular functions of endothelial cells by attenuating protein S-nitrosylation.

Cardiovascular side effects of broadly used chemotherapeutic drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) among cancer survivors are well established. Nitric oxide (NO) is known to protect cardiovascular tissues under conditions of stress. NO can act through cyclic guanosine monophosphate (cGMP)-dependent and -independent pathways. Particularly, the S-nitrosylation of SH-groups in a protein by NO falls under cGMP-independent effects of NO. TC, CP, and EP are hypothesized as interfering with cellular protein S-nitrosylation, which, in turn, may lead to endothelial dysfunctions. The results show that all three drugs attenuate nitrosylated proteins in endothelial cells. A significant reduction in endogenous S-nitrosylated proteins was revealed by Saville-Griess assay, immunofluorescence and western blot. Incubation with the drugs causes a reduction in endothelial migration, vasodilation and tube formation, while the addition of S-nitrosoglutathione (GSNO) has a reversal of this effect. In conclusion, results indicate the possibility of decreased cellular nitrosothiols as being one of the reasons for endothelial dysfunctions under TC, CP and EP treatment. Identification of the down-regulated S-nitrosylated proteins so as to correlate their implications on fundamental vascular functions could be an interesting phenomenon.

Enhanced butanol production from cassava with Clostridium acetobutylicum by genome shuffling.

To obtain strains exhibiting high levels of solvent tolerance and butanol production, wild type strains of Clostridium acetobutylicum butanol-producing strain GX01 and Lactobacillus mucosae butanol-tolerant strain M26 were subjected to mutagenesis combining N-methyl-N-nitro-N-nitrosoguanidine induction with genome shuffling. After four successive rounds of genome shuffling, the C. acetobutylicum shuffled strain GS4-3 showing greater levels of fermentation performances (such as secreting a higher level of amylase, improving the thermal stability, and possessing greater environmental robustness) compared to the wild type strains was isolated. As a result, after optimization of culture conditions, mutant GS4-3 produced 32.6 g/L of total solvent, 20.1 g/L of butanol production, and 0.35 g/L/h of butanol productivity, which were, respectively, increased by 23.5, 23.3, and 40.0 % than the wild-type strain GX01, in a 10 L bioreactor. The enhanced production of butanol and tolerance of solvent of mutant associated with GS4-3 make it promising for acetone/butanol/ethanol fermentation from cassava (Manihot esculenta).

Endogenic oxidative stress response contributes to glutathione over-accumulation in mutant Saccharomyces cerevisiae Y518.

Mechanisms of glutathione (GSH) over-accumulation in mutant Saccharomyces cerevisiae Y518 screened by ultraviolet and nitrosoguanidine-induced random mutagenesis were studied. Y518 accumulated higher levels of GSH and L-cysteine than its wild-type strain. RNA-Seq and pathway enrichment analysis indicated a difference in the expression of key genes involved in cysteine production, the GSH biosynthesis pathway, and antioxidation processes. GSH1, MET17, CYS4, GPX2, CTT1, TRX2, and SOD1 and the transcriptional activators SKN7 and YAP1 were up-regulated in the mutant. Moreover, Y518 showed a dysfunctional respiratory chain resulting from dramatically weakened activity of complex III and significant elevation of intracellular reactive oxygen species (ROS) levels. The supplementation of antimycin A in the culture of the parent strain showed equivalent changes of ROS and GSH level. This study indicates that defective complex III prompts abundant endogenic ROS generation, which triggers an oxidative stress response and upregulation of gene expression associated with GSH biosynthesis. This finding may be helpful for developing new strategies for GSH fermentation process optimization or metabolic engineering.

Acetone, butanol, and ethanol production from cane molasses using Clostridium beijerinckii mutant obtained by combined low-energy ion beam implantation and N-methyl-N-nitro-N-nitrosoguanidine induction.

In order to obtain mutant strains showing higher solvent tolerance and butanol production than those of wild-type strains, the butanol-producing strain Clostridium beijerinckii L175 was subjected to mutagenesis using a combined method of low-energy ion beam implantation and N-methyl-N-nitro-N-nitrosoguanidine induction. With this effort, mutant strain MUT3 was isolated. When it was used for butanol fermentation in P2 medium, the production of butanol was 15.8±0.7 g/L 46% higher than the wild-type strain. Furthermore, after optimization of butanol production from cane molasses with MUT3, the maximum butanol production of 14.9±0.5 g/L were obtained in crew-capped bottles. When ABE production by MUT3 was carried out in a bioreactor, the production of butanol and total solvent were 15.1±0.8 g/L and 22.1±0.9 g/L, respectively. The remarkable butanol production and solvent tolerance of MUT3 make it promising for butanol production from cane molasses.

Multiple induced mutagenesis for improvement of ethanol production by Kluyveromyces marxianus.

Kluyveromyces marxianus GX-15 was mutated multiple times by alternately treatment with UV irradiation and NTG for two cycles. Four mutant strains with improved ethanol yield were obtained. The maximum ethanol concentration, ethanol yield coefficient and theoretical ethanol yield of the best mutant strain, GX-UN120, was 69 g/l, 0.46 g/g and 91%, respectively, when fermenting 150 g glucose/l at 40°C. The corresponding values for GX-15 were 58 g/l, 0.39 g/g and 76%, respectively. GX-UN120 grew well in 11% (v/v) of ethanol, while GX-15 could not grow when ethanol was greater than 8% (v/v).

Identification of aldehyde oxidase as the neonicotinoid nitroreductase.

Imidacloprid (IMI), the prototypical neonicotinoid insecticide, is used worldwide for crop protection and flea control on pets. It is both oxidatively metabolized by cytochrome P450 enzymes and reduced at the nitroguanidine moiety by a previously unidentified cytosolic "neonicotinoid nitroreductase", the subject of this investigation. Two major metabolites are detected on incubation of IMI with rabbit liver cytosol: the nitrosoguanidine (IMI-NO) and the aminoguanidine (IMI-NH2). Three lines of evidence identify the molybdo-flavoenzyme aldehyde oxidase (AOX, EC 1.2.3.1) as the neonicotinoid nitroreductase. First, classical AOX electron donor substrates (benzaldehyde, 2-hydroxypyrimidine, and N-methylnicotinamide) dramatically increase the rate of formation of IMI metabolites. Allopurinol and diquat are also effective electron donors, while NADPH and xanthine are not. Second, AOX inhibitors (potassium cyanide, menadione, and promethazine) inhibit metabolite formation when N-methylnicotinamide is utilized as an electron donor. Without the addition of an electron donor, rabbit liver cytosol reduces IMI only to IMI-NO at a slow rate. This reduction is also inhibited by potassium cyanide, menadione, and promethazine, as well as by additional AOX inhibitors, cimetidine and chlorpromazine. Finally, IMI nitroreduction by AOX is sensitive to an aerobic atmosphere, but to a much lesser extent than cytochrome P450 2D6. Large species differences are observed in the IMI nitroreductive activity of liver cytosol. While rabbit and monkey (Cynomolgus) give the highest levels of total metabolite formation, human, mouse, cow, and rat also metabolize IMI rapidly. In contrast, dog, cat, and chicken liver cytosols do not reduce IMI at appreciable rates. AOX, as a neonicotinoid nitroreductase, may limit the persistence of IMI, and possibly other neonicotinoids, in mammals.

Evolution of differential substrate specificities in Mu class glutathione transferases probed by DNA shuffling.

A library of variant enzymes was created by combined shuffling of the DNA encoding the human Mu class glutathione transferases GST M1-1 and GST M2-2. The parental GSTs are 84 % sequence identical at the protein level, but their specific activities with the substrates aminochrome and 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG) differ by more than 100-fold. Aminochrome is of particular interest as an oxidation product of dopamine and of possible significance in the etiology of Parkinson's disease, and cyanoDMNG is a model for genotoxic and potentially carcinogenic nitroso compounds. GST M2-2 has at least two orders of magnitude higher catalytic activity with both of the substrates than any of the other known GSTs, including GST M1-1. The DNA library of variant Mu class GST sequences contained "mosaic" structures composed of alternating segments of both parental sequences. All clones contained the 5'-end of a GST M1-1 clone optimized for high-level expression in Escherichia coli. The remainder of the sequences derived from segments of GST M2-2 and GST M1-1 DNA. All of the clones analyzed contained between two and seven distinct DNA segments. In addition, each clone contained an average of approximately one point mutation. None of the library clones analyzed was identical with either of the two parental structures. Variant GST sequences were expressed in E. coli, and their enzymatic activities with aminochrome, cyanoDMNG, and 1-chloro-2,4-dinitrobenzene (CDNB) were determined in bacterial lysates. Such screening of more than 70 clones demonstrated a continuous range of activities covering at least two orders of magnitude for each of the substrates. For a given clone, the activities with aminochrome and cyanoDMNG, in spite of their different chemistries, were clearly correlated, whereas no strong correlation was found with CDNB. This functional correlation suggests a common structural basis for the enzymatic mechanisms for conjugation of aminochrome and denitrosation of cyanoDMNG. From an evolutionary perspective, the results show that recombination of segments from homologous proteins gives rise to a large proportion of functionally competent proteins with a range of activities. The data support the proposal that natural evolution of protein functions may involve recombination of DNA segments followed by selection for advantageous functional properties of the resulting proteins. Clearly, the same approach can be utilized in the engineering of proteins displaying novel functions by in vitro evolution.

Enzymic denitrosation of 1,3-dimethyl-2-cyano-1-nitrosoguanidine in rat liver cytosol and the fate of the immediate product S-nitrosoglutathione.

The tumorigenicity of certain N-nitrosoguanidinium compounds is limited, in rodents, by the propensity of these agents to be detoxified by denitrosation. Previous studies have revealed that rodent glutathione transferase isoenzymes are capable of catalyzing this process, generating exclusively the denitrosated guanidinium compound and S-nitrosoglutathione (GSNO). Experiments considering the denitrosation of 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG) in rat liver cytosol incubates are reported, with emphasis on the fate of GSNO. Incubates composed with equimolar CyanoDMNG and reduced glutathione (GSH) effected 100% denitrosation; the GSNO yield was less than expected as was the quantity of GSH consumed. When the anticipated 100% yield concentration of GSNO was applied to cytosol incubates, 20-40% of it rapidly disappeared. Nitrosated protein thiols accounted for 35% of the NO moiety released, nitrite ion 30%, and nitric oxide production was detectable. Concomitant with GSNO loss, GSH and oxidized glutathione (GSSG) were generated in yields similar to those detected in the CyanoDMNG/GSH incubates. Thus, the fate of GSNO in cytosol determines the yields of glutathione-based products, and the stoichiometry of the glutathione transferase reaction is demonstrated. In incubates composed with equimolar CyanoDMNG, GSH, and NADPH, denitrosation was again 100%, but GSNO yields were very low and residual GSH increased. Inclusion of NADPH in incubates containing the anticipated 100% yield concentration of GSNO resulted in rapid GSNO degradation, producing GSH and a detected but unidentified product; S-nitrosated protein, nitrite, and nitrate yields were minimal, nitric oxide production was abolished, and incubate response to a mercuric chloride/azo dye assay approached zero. The fate of the NO moiety consequent to this GSNO catabolism is presently unknown.

Denitrosation of 1,3-dimethyl-2-cyano-1-nitrosoguanidine in rat primary hepatocyte cultures.

N-Nitrosoguanidines are potential carcinogens. However, the toxicity of these agents is attenuated significantly in laboratory rodents by processes that remove the nitroso group to generate the relatively innocuous parent guanidinium compound. The denitrosation of 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG) mediated by rat hepatocytes in primary culture was investigated. At concentrations < or = 200 microM, applied CyanoDMNG was converted efficiently to 1,3-dimethyl-2-cyanoguanidine (CyanoDMG). In trials using 50 microM CyanoDMNG (5 mL dosing solutions), it was demonstrated that hepatocytes are capable of denitrosating a 40 microM concentration of the applied compound with little change in the total or oxidized glutathione levels. The process was inhibited by coincidently applied ethacrynic acid, a glutathione transferase inhibitor. Reduction of hepatocyte glutathione to 20% of control levels by buthionine sulfoximine pretreatment had little effect on CyanoDMG production; total depletion of cytosolic glutathione by diethyl maleate pretreatment arrested CyanoDMNG processing. Hepatocyte-mediated CyanoDMNG denitrosation did not generate nitrite; nitrate yields were 10% relative to the CyanoDMG produced. The mercuric chloride/azo dye response of cultures lysed at times during 50 microM CyanoDMNG processing indicated intact CyanoDMNG as the only dye-sensitive material present. At applied CyanoDMNG > 100 microM, S-nitrosoglutathione (GSNO) yields were detectable; 4 microM GSNO was generated (concentration in 5 mL lysates) and maintained during 60 min at the 200 microM CyanoDMNG treatment level; this yield decayed if CyanoDMNG was withdrawn. Based on these and previous findings, it is hypothesized that CyanoDMNG is converted to CyanoDMG and GSNO by glutathione transferases and that GSNO is catabolized to eventually regenerate reduced glutathione. The fate of most of the NO moiety released remains to be determined.

Microsomally-mediated denitrosation of nitrosoguanidinium compounds.

A major metabolic fate of 1-methyl-2-nitro-1-nitrosoguanidine (MNNG) and nitrosocimetidine (NC) in rodents is denitrosation to generate the unmodified, parent guanidinium compound. MNNG is a potent, locally-acting carcinogen. NC is the nitrosated derivative of cimetidine, an important clinical drug administered orally for the treatment of stomach ulcers. Contrary to expectations based on the results of various short-term in vitro tests for carcinogenic potential, NC is not a carcinogen when administered to rats or mice. Rat liver microsomal enzymes have been found to be capable of catalyzing the denitrosation of MNNG, NC and an NC analog, 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG) in an NADPH-dependent reaction. The denitrosated guanidinium compound generated accounts for 50-70% of the nitroso compound metabolized in a microsomal incubate; nitrite is generated with a yield which represents 40-60% of the guanidinium compound produced. The cytochrome P450 inhibitors metyrapone, n-octylamine, 1-n-hexylimidazole and ellipticine inhibit the conversion of CyanoDMNG to 1,3-dimethyl-2-cyanoguanidine (Cyano-DMG) and nitrite. Microsomal NADPH-cytochrome c reductase activity is not perturbed by this series of organic compound inhibitors. Diethyl maleate at high concentrations weakly stimulates the reaction. The rates of production of the CyanoDMNG degradation products CyanoDMG, nitrite and nitrate are markedly diminished in nitrogen-saturated and in carbon dioxide-saturated microsomal incubates. Preincubating microsomes for 1 h at 37 degrees C prior to substrate and NADPH addition has no effect on the denitrosation activity. Kinetic analysis of the conversion of CyanoDMNG to CyanoDMG indicates a Km of 1.0 mM and a Vmax of 2.7 nmol/min/mg protein. Microsomes isolated from rats pretreated with the cytochrome P450 inducers pyrazole or phenobarbital show enhanced denitrosation activity. The denitrosation capacity of hamster liver microsomes is similar to that observed for rat microsomes.

Rat, mouse and hamster isozyme specificity in the glutathione transferase-mediated denitrosation of nitrosoguanidinium compounds.

The major isozymes from affinity column-purified glutathione transferases isolated from Sprague-Dawley rat liver, kidney, and testis cytosol and also from BALB/c mouse and Syrian golden hamster liver cytosol have been resolved by chromatofocusing and tested for their ability to denitrosate and thus detoxicate the DNA-methylating agents and potential carcinogens nitrosocimetidine and 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG). The isozymes have been kinetically characterized using a battery of substrates permitting, in the rat and mouse cases, subunit composition identification. It has been found that the rat and mouse isozymes belonging to the mu class are uniquely and highly active in the denitrosation of nitrosocimetidine and CyanoDMNG. A specific set of hamster glutathione transferase isozymes were also found to be active in these reactions. We have identified the reaction products produced by the rat liver 3-4 isozyme activity. The glutathione transferase-mediated degradations of 1-methyl-2-nitro-1-nitrosoguanidine and CyanoDMNG generate one molecule of S-nitrosoglutathione per molecule of denitrosated guanidinium compound produced. In the CyanoDMNG incubations essentially all degradation was via denitrosation; nitrite and glutathione disulfide were minor products. In the 1-methyl-2-nitro-1-nitrosoguanidine case nonenzymic degradation of the nitroso compound in the presence of reduced glutathione was evident but little of this decomposition produced S-nitrosoglutathione or 1-methyl-2-nitroguanidine. In the presence of rat transferase 3-4 isozyme, glutathione-dependent 1-methyl-2-nitro-1-nitrosoguanidine degradation was shifted markedly towards denitrosation with the concomitant production of S-nitrosoglutathione.

The binding of benzo(alpha)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine to subnuclear fractions of AKR mouse embryo cells in culture.

The marked localization of a carcinogenic polycyclic aromatic hydrocarbon, benzo(alpha)pyrene, and its metabolites and a carcinogenic alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, to a specific subnuclear fraction (fraction I) from AKR-2B mouse embryo cells in culture is described. Fraction I is isolated by sucrose gradient centrifugation of sheared nuclei from cells exposed to the carcinogens. The association of tritiated benzo(alpha)-pyrene to fraction I consisted of loosely associated radioactivity which is extractable by organic solvents, and of tightly bound (termed "covalently" bound) radioactivity which is not extractable by organic solvents. Increases in the extent of metabolism of benzo(alpha)pyrene and in the amount of "covalently" bound radioactivity occur with increasing periods of incubation of the cells with the labelled carcinogen. This observation, together with the fact that these increases are dramatically reduced by inhibiting polycyclic aromatic hydrocarbon metabolism (using the inhibitor 7,8-benzo-flavone), suggests that a time-dependent metabolism of benzo(alpha)pyrene is required for "covalent" binding to muclear material. Data are presented suggesting that a two-step reaction may be involved in the binding of benzo(alpha)pyrene to subnuclear macromolecules. The fraction I localization of such structurally diverse chemical carcinogens as benzo(alpha)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine suggests that this fraction may localize all species of chemical carcinogens and that this localization may be involved in the chemically induced malignant transformation of cells.

Tissue distribution and mode of DNA methylation in mice by methyl methanesulphonate and N-methyl-N' -nitro-N-nitrosoguanidine: lack of thymic lymphoma induction and low extent of methylation of target tissue DNA at 0-6 of guanine.

The methylating agents methyl methanesulphonate (MMS) and N-methyl N'-nitro-N-nitrosoguanidine (MNNG), administered by single i.p. injection in mice failed to yield thymic lymphoma at doses around 60% of the LD50 values, in contrast to MNUA which gives a high yield of tumours by this route. Comparison of the tissue distribution and mode of DNA methylation by these agents showed a positive correlation with ability to methylate the 0-6 atom of guanine in DNA of the target tissues thymus and bone marrow and tumorigeneis. MMS gave a low yield of this product due to its relatively low Sn1 reactivity but was able to methylate DNA extensively at other sites in the target tissues and other organs examined. MNNG despite its ability to methylate 0-6 of guanine in DNA in vitro to the same relative extent as the potent carcinogen MNUA, methylated DNA of thymus and bone marrow to a very small extent in vivo but was able to methylate DNA in certain other tissues nearer the site of i.p. injection. These findings contrast with the general relatively extensive methylation of 0-6 of guanine in DNA of the target tissues and other organs by N-methyl-N-nitrosourea (MNUA).

Mutant enrichment in the colonial alga, Eudorina elegans.

An enrichment procedure has been developed that results in at least a 200 X increase in mutation frequency in the colonial alga, Eudorina elegans. A period of nitrogen starvation followed by treatment with 8-azaguanine results in the death of wild-type cells and the maintenance of mutants. N'-nitro-N-nitro-soguanidine-induced acetate, p-aminobenzoic acid and reduced nitrogen requiring mutants have been isolated by this procedure.