Prophage Induction in Lysogenic Escherichia coli with Simple Hydroxylamine and Hydrazine Compounds

The prophage-inducing capability of hydroxylamine sulfate and 36 of its derivatives, and of hydrazine dihydrochloride and dihydrazine sulfate and 43 of their derivatives, was determined in Escherichia coli W1709 (lambda). Maximal nontoxic concentrations up to 1 mg/ml were tested. Hydroxylamine sulfate was active at 2.5 mug/ml and the following 17 derivatives were active at concentrations ranging up to 500 mug/ml: alpha-naphthylhydroxylamine, N-hydroxy-2-aminofluorene, oxamyl hydroxamic acid, O-carbamoyl hydroxylamine (isohydroxyurea), N-hydroxyurethane, N-methylhydroxylamine HCl, salicylhydroxamic acid, oxalohydroxamic acid, methoxyamine HCl, ethoxyamine HCl, N, N-diethylhydroxylamine oxalate, formaldoxime, formamidoxime, acetohydroxamic acid, acetaldoxime, acetone oxime, and hydroxyguanidine sulfate. Hydrazine dihydrochloride and dihydrazine sulfate were effective inducers at 5.0 and 2.5 mug/ml, respectively, and the following nine derivatives of them were active at concentrations ranging up to 500 mug/ml: phthalic acid hydrazide, phenylhydrazine HCl, p-nitrophenylhydrazine, p-chlorophenylhydrazine HCl, formylhydrazine, carbohydrazide, semicarbazide HCl, 1-methyl-1-phenyl-hydrazine sulfate, and acetic acid hydrazide. Nineteen hydroxylamine and 34 hydrazine derivatives were ineffective as inducers. Application of the prophage-induction system as a tool for detection of responsive hydroxylamino and hydrazino compounds which may be potential toxicological hazards in the environment is discussed.     

Mechanisms of inhibition of N-nitroso compounds-induced mutagenicity

In this review we describe the mechanisms of the inhibitory effects of various chemical agents towards the mutagenicity of N-nitroso compounds, including direct-acting mutagens such as N-nitroso derivatives of alkylureas, alkylnitroguanidines and alkylurethanes, and promutagenic nitrosamines. Possible mechanisms by which the inhibitors may exert their effects outside and inside the target cells include chemical and enzymatic deactivation of the mutagen, inhibition of metabolic activation of nitrosamines, scavenging mutagenic products, inhibition of cellular uptake, induction of detoxifying mechanisms, protecting nucleophilic centers in DNA and modulating DNA repair.     

Photolysis of nitrosamines and nitrosamides at neutral pH: a spin-trap study

A model system has been used to study the types of radicals formed on denitrosation of N-nitroso compounds. Free radicals were formed at room temperature (22 degrees-23 degrees C) and neutral pH by photolytic cleavage of N-nitroso bonds and were partially characterized following their addition to the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenyl-nitrone (PBN). Carbon-centered radical adducts were obtained during nitrosamine photolysis and nitrogen-centered radical adducts during nitrosamide photolysis. Since both the nitrosamines and nitrosamides initially form nitrogen-centered radicals on photolysis, a secondary reaction or rearrangement must occur after initial N-nitroso bond cleavage in the nitrosamines. Mechanisms are proposed to account for these results.     

Repair of DNA alkylation adducts in mammalian cells

Carcinogenic alkylating agents, including nitrosamines, are able to alkylate DNA at various sites. This review presents evidence of the high degree of specificity in the type of DNA damage induced by various N-nitroso compounds and in the DNA repair processes among tissues or cells of different species. The O6-alkylguanine DNA alkyltransferase activity in various human and rodent tissues is discussed as well as the detection of O6-methylguanine in human DNA, using monoclonal antibodies and radioimmunoassay. The relevance of these findings to the mechanisms of cancer induction by nitrosamines is discussed.     

Nitrite, nitrosamines, and cancer.

Carcinogenic N-nitroso compounds are formed from the reaction of naturally-occurring amines and nitrites that may be added to foods or produced by bacterial reduction of nitrate. N-Nitroso compounds can be produced during processing, storage and preparation of foods and in the mammalian stomach. Factors that influence the rates of nitrosation reactions include pH, temperature, catalysts, and inhibitors. Predictions of the extent of nitrosation are complicated by these factors and ultimately the amounts and types of N-nitroso compounds present must be determined by direct analysis. Methods for detection and estimation of volatile nitrosamines are available and low levels (parts per billion) have been found in some cured meat and fish products. General methods for detection of all N-nitroso compounds are not available yet, but are under development. Evaluation of the risk to human populations from these compounds is difficult in the absence of more comprehensive data on their environmental distribution.     

Mutagenicity and cytotoxicity of congeners of two classes of nitroso compounds in Chinese hamster ovary cells

The induction of mutation by certain nitrosamidines and nitrosamides has been quantitated utilizing the hypoxanthine--guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells. Dose--response relationships for cytotoxicity and mutagenicity are presented for N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-butyl-N-nitrosourea (BNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Based on the concentration of each agent required to kill 90% of the cells, the following order of cytotoxicity was observed: MNNG greater than ENNG greater than MNU greater than ENU greater than BNU. This is the same order of potency as observed for mutation induction per unit concentration of mutagen.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC Working Paper No. 15/6. Effect of ethanol on nitrosamine metabolism and distribution. Implications for the role of nitrosamines in human cancer and for the influence of alcohol consumption on cancer incidence

Alcohol consumption is associated with an increase in the incidence of cancers of several sites, including oesophagus, larynx and mouth. The mechanism of the induction of cancer by alcohol is not clear. Humans are exposed to a variety of carcinogenic N-nitroso compounds. Ethanol changes the pharmacokinetics of nitrosamines in rats particularly by decreasing the ability of the liver to metabolize them. A hypothesis is put forward that the influence of alcohol on human cancer is mediated by its effect on the metabolism and distribution of nitrosamines from the diet, from tobacco smoke and from endogenous synthesis.     

Deuterium isotope effects in mutagenesis by nitroso compounds.

Nitrosamines which have deuterium instead of hydrogen in the position alpha to the nitroso group have been reported to have reduced activity in carcinogenicity tests. This result implies that cleavage of a carbon--hydrogen bond is a limiting step in the reaction mechanism leading to tumor formation. Mutagenicity tests were undertaken with nitrosamines, which require metabolic activation, and with nitrosamides, which are directly acting mutagens, to determine the effect of deuterium substitution on the activity of each type of compound. Two nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine and methylnitrosourea) and three nitrosamines (dimethylnitrosamine, nitrosomorpholine, and dinitrosopiperazine) and their deuterium-containing analogs were tested for reversion of a nonsense mutation in the tyr locus of Escherichia coli WU 3610 (tyr-, leu-). Nitrosamines activated by rat-liver microsomes, but not nitrosamides, were less active as mutagens when the deuterium atom was present. The results suggest that the metabolic activation of nitrosamines to a mutagenic species involves the loss of hydrogen, a reaction which the nitrosamides, in the absence of enzyme, do not undergo.     

Characterization of a CHO variant in respect to alkylating agent-induced biological effects and DNA repair

From the Chinese hamster ovary line CHO-9 a resistant variant, Cl 3, was isolated after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cl 3 cells were much more resistant to the cytotoxic effects of MNNG (D10 of 1.8 microgram/ml MNNG as compared to 0.23 microgram/ml for parental line) and other methylating N-nitroso compounds, but they had the same sensitivity to various other alkylating agents. MNNG was equally effective in sensitive parent line and resistant variant in inducing sister-chromatid exchanges (SCEs) and mutations to 6-thioguanine resistance. The increased resistance of Cl 3 was not due to reduced cellular uptake of MNNG, to a more efficient repair of methylated purine bases, or to differences in MNNG-induced inhibition of DNA synthesis. It is concluded that the resistant variant has some unknown tolerance mechanism which alters the cytotoxic, but not the SCE- and mutation-inducing effects of methylating N-nitroso compounds.     

Synthesis and evaluation of the permeability transition inhibitory characteristics of paramagnetic and diamagnetic amiodarone derivatives

Several amiodarone analogues were synthesized varying the 2-substituent on the benzofuran ring and diethylaminoethyl side chain of phenolether by introducing 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole and 1,2,5,6-tetrahydropyridine nitroxides or their amino or hydroxylamino precursors. The new compounds were screened on isolated mitochondria and perfused heart and their toxicity was evaluated on WRL-68 liver cells and H9C2 cardiomyocytes. Most of the newly synthesized derivatives exerted uncoupling effect on the mitochondrial oxidative phosphorilation at higher concentrations, compared to amiodarone and one of the modified amiodarone analogues showed an effect similar to that of amiodarone on the mitochondrial permeability transition and on restoring of mitochondrial high-energy phosphate metabolites in perfused hearts. This amiodarone analogue can be new leading compound among the experimental amiodarone analogues with the same or enhanced efficiency of amiodarone, but with less side effects.     

Metabolic activation capabilities of S9 and hepatocytes from uninduced rats to convert carcinogenic N-nitrosamines to mutagens

6 carcinogenic nitrosamines were studied in Salmonella typhimurium TA1535 after activation by S9 and by hepatocytes. All nitrosamines were activated by S9 from induced rats, regardless of their organotropy. The hepatocarcinogenic nitrosamines (N-nitrosodimethylamine, NDMA; N-nitrosodiethylamine, NDEA; N-nitrosomorpholine, NM and N-nitrosodibutylamine, NDBA) were activated to mutagens by S9 and by hepatocytes both derived from noninduced rat livers, NDMA and NM inducing more his+ revertants in the presence of hepatocytes. The oesophageal carcinogenic nitrosamine N-nitrosomethylbenzylamine (NMBeA) and bladder organotrophic N-nitroso(4-hydroxybutyl)butylamine(NBBOH) were neither converted by liver preparations of uninduced rats into mutagenic intermediates nor by hepatocytes. This study indicates that isolated cells derived from untreated animals may be better suited to study liver specific activation in vitro than disrupted subcellular metabolizing systems from induced animals.     

Stereochemical effects in the metabolic activation of nitrosopiperidines: correlations with genotoxicity

The genotoxicity of nitrosopiperidine and six alkyl derivatives was studied by use of the Ames tester strains TA100 and TA1535 in the pre-incubation method. Among the compounds investigated, those exhibiting genotoxic activity under the experimental conditions employed were only genotoxic in the presence of S9 mix (10%). The results obtained were correlated with models of the metabolic activation of nitrosamines in an attempt to rationalize the genotoxicity of these compounds. The results show that the presence of substituents in the nitrosopiperidine molecule may be one of the modulating factors affecting the genotoxicity of these cyclic nitrosamines, and may help provide some chemical clues for the identification of risk compounds from among a large group of structurally related molecules.     

N-Nitroso compounds in the diet.

N-Nitroso compounds were known almost 40 years ago to be present in food treated with sodium nitrite, which made fish meal hepatotoxic to animals through formation of nitrosodimethylamine (NDMA). Since that time, N-nitroso compounds have been shown in animal experiments to be the most broadly acting and the most potent group of carcinogens. The key role of nitrite and nitrogen oxides in forming N-nitroso compounds by interaction with secondary and tertiary amino compounds has led to the examination worldwide of foods for the presence of N-nitroso compounds, which have been found almost exclusively in those foods containing nitrite or which have become exposed to nitrogen oxides. Among these are cured meats, especially bacon-and especially when cooked; concentrations of 100 micrograms kg(-1) have been found or, more usually, near 10 micrograms kg(-1). This would correspond to consumption of 1 microgram of NDMA in a 100-g portion. Much higher concentrations of NDMA (but lower ones of other nitrosamines) have been found in Japanese smoked and cured fish (more than 100 micrograms kg(-1)). Beer is one source of NDMA, in which as much as 70 micrograms l(-1) has been reported in some types of German beer, although usual levels are much lower (10 or 5 micrograms l(-1)); this could mean a considerable intake for a heavy beer drinker of several liters per day. Levels of nitrosamines have been declining during the past three decades, concurrent with a lowering of the nitrite used in food and greater control of exposure of malt to nitrogen oxides in beer making. There have been declines of N-nitroso compound concentrations in many foods during the past two decades. The small amounts of nitrosamines in food are nonetheless significant because of the possibility-even likelihood-that humans are more sensitive to these carcinogens than are laboratory rodents. Although it is probable that alkylnitrosamides (which induce brain tumors in rodents) are present in cured meats and other potentially nitrosated products in spite of much searching, there has been only limited indirect evidence of their presence.     

Amino and hydrazino alkyl benzoates as derivatizing agents for the separation and mass spectrometric analysis of oligosaccharides from bacterial lipooligosaccharides

In an attempt to develop more sensitive and versatile methods for the structure analysis of oligosaccharides derived from lipooligosaccharides (LOS) of gram-negative bacteria, amino and hydrazino alkyl benzoate derivatives were prepared. These oligosaccharide derivatives were separated by HPLC and then analyzed by liquid secondary ion mass spectrometry (LSIMS). Both the amino and hydrazino alkyl benzoates react with the free reducing termini of acid-treated LOS, increasing the hydrophobicity of the released oligosaccharides and allowing them to be separated by reverse-phase HPLC. In addition, these oligosaccharide derivatives now contain a sensitive uv chromophore for subsequent peak detection and improve the quality of the LSIMS spectra compared to underivatized oligosaccharides. However, the amino alkyl benzoates reacted poorly compared to the analogous hydrazino alkyl benzoates with 3-deoxy-manno-2-keto octulosonic acid (KDO), and oligosaccharides with KDO at the reducing terminus, especially when the oligosaccharide also contained phosphoethanolamine. Derivatization with the hydrazino compounds can be carried out quickly and under mild conditions using a minimal amount of reagent, and is therefore suitable for microscale analyses. The chromatographic and mass spectrometric characteristics of these derivatives make them excellent alternatives to permethylation and peracetylation techniques for the structural analysis of complex bacterial oligosaccharides derived from glycolipids.     

Carcinogenesis and nucleic acid alkylation by some oxygenated nitrosamines in rats and hamsters

A comparison has been made of the carcinogenic effects of nitroso-2,6-dimethylmorpholine and several hydroxylated acyclic nitrosodialkylamines derived from it or related to it in rats and Syrian hamsters. In rats nitrosodimethylmorpholine was the most potent, inducing mainly esophageal tumors. Nitrosodiethanolamine was the weakest of the five nitrosamines in both rats and hamsters. Tumors of the pancreas ducts were induced by four of the five compounds, but only in hamsters, and esophageal tumors appeared only in rats. Most of the nitrosamines induced tumors of liver and lung in both rats and hamsters. A study of alkylation of nucleic acids of the liver following treatment of rats and hamsters with the radiolabeled nitrosamines showed that nitrosodiethanolamine alkylated liver nucleic acids in rats to only a very small extent. The other four nitrosamines all gave rise to 7-methylation and O6-methylation of guanine residues in DNA of hamster liver and all but nitrosodimethylmorpholine in rat liver DNA, which corresponded quite well with the induction of liver tumors in the two species. Quantitatively, however, there was not a good correlation between liver DNA alkylation and the potency of the nitrosamine in inducing tumors.     

Determination of isoniazid and its hydrazino metabolites, acetylisoniazid, acetylhydrazine, and diacetylhydrazine in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric assay for isoniazid and its hydrazino metabolites in human plasma was developed. The trimethylsilyl derivatives of diacetylhydrazine and acetylisoniazid and of the benzaldehyde hydrazones of acetylhydrazine and isoniazid were separated on a 1% OV-17 column and quantitated by single ion monitoring using a LKB 9000 mass spectrometer. Deuterated analogues served as internal standards. The method is well suited for the determination of the hepatotoxic hydrazino metabolites of isoniazid in human plasma following an oral therapeutic dose of isoniazid.     

Colorimetric determination of N-hydroxylated metabolites in microsomal studies

A colorimetric method is described which can be used for the routine determination of primary and secondary N-hydroxylamino compounds in drug metabolism studies using microsomal preparations. The assay is carried out on the supernatant obtained after protein precipitation of the incubation mixture. The method is based on the reduction of ferric ion by the hydroxylamino moiety with the resulting ferrous ion being quantitated by coupling with 2,4,6-tripyridyl-s-triazine to form a purple color with a maximum absorbance at 595 nm. The method is specific to primary and secondary hydroxylamino compounds and calibration curves can be obtained in the range of concentrations of 1 to 20 μg/ml. Hydroxamic acid, amido, amino, phenolic, nitro, nitroso, oxime, nitrone, aldehyde, and ketone compounds do not produce any color reaction when analyzed by the same method. The method is simple, rapid, and many samples can be analyzed in a short period of time.     

Influence of glucagon, 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and triamcinolone on the arginine synthetase system in perinatal rat liver

1. The administration of triamcinolone (19-190mug/animal) to postnatal rats increased the arginine synthetase system activity 1.2-2.5-fold above control values 24h after exposure to the hormone. Cortisol (hydrocortisone), however, increased the arginine synthetase system activity only when larger (190mug/animal) or repeated daily doses were given. Glucagon (100mug/animal) stimulated arginine synthetase system activity only after the second postnatal day. None of these agents increased the activity in 19.5-21.5-day foetuses after intrauterine administration. 2. The viability of foetal rat liver explants maintained in organ culture for up to 54h was validated both by ultramicroscopic examination and by incorporation of radioactive leucine and orotic acid. 3. In organ cultures of foetal rat liver explants (18.5 days to term), triamcinolone (20mug/ml of medium) evoked a 2.8-4.3-fold increase after 24h of incubation. This increase was completely inhibited by actinomycin D (25mug/ml) or cycloheximide (10mug/ml). Cortisol (5-50mug/ml) or glucagon (0.067-67mug/ml) also increased the arginine synthetase system activity above the respective control values, but there was no increase in activity with insulin (0.05-0.25i.u./ml). 4. Maximum concentrations of glucagon (67mug/ml), dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) (0.1mm) and triamcinolone (20mug/ml) incubated for 24h with foetal rat liver explants each produced between a two-and three-fold increase in the activity of the arginine synthetase system. Combinations of maximum amounts of glucagon and the cyclic nucleotide did not produce a greater effect than either agent alone. However, the combination of dibutyryl cyclic AMP with triamcinolone appeared to produce somewhat less than additive effects. 5. The effects of the cyclic nucleotide and triamcinolone were evident after 12h of incubation and increased steadily throughout the 24h of observation. This time-course of increased enzyme activity is very much slower than that reported for the induction of other enzymes in explant cultures of foetal rat liver.     

Rapid determination of artemisinin and related analogues using high-performance liquid chromatography and an evaporative light scattering detector

Artemisinin and its analogues are a class of compounds of current interest in the treatment of drug-resistant malaria. These antimalarials are preferentially taken up into malaria infected erythrocytes as compared to uninfected erythrocytes, a fact that may represent an important parameter in drug potency. Numerous methods for the analysis of specific artemisinin analogues have been developed, but most are not widely adaptable to a large range of analogues. In this paper we describe a high-performance liquid chromatographic method developed and validated for artemisinin and several analogues of artemisinin using a readily available evaporative light scattering detector. This quantitation method was found to be straight forward, rapid, inexpensive and reproducible. Standard calibration curves constructed for six artemisinin compounds were linear with the detection limit determined between 6 and 60 ng. The intra- and inter-day accuracy were found to be 2.75% and 4.15%, respectively with less than 3% variation in precision. The validated assay was applied to a mixture of artemisinin derivatives, where they were easily separated and quantitated.     

Induction of a Biopolyester Hydrolase (Cutinase) by Low Levels of Cutin Monomers in Fusarium solani f. sp. pisi

Cutin hydrolysate induced the production of an extracellular cutinase by glucose-grown Fusarium solani f. sp. pisi. The rate of production depended on the amount of cutin hydrolysate added up to 80 mug/ml, and saturation was attained at this level. Glucose was found to be a repressor of cutinase production. A radial immunodiffusion assay for cutinase was developed, and the induction of cutinase by cutin hydrolysate was confirmed by this direct assay. When cutinase was induced by cutin hydrolysate, exogenous labeled phenylalanine was incorporated into cutinase, which was shown to be the major (>70%) protein in the extracellular fluid. Induction of cutinase by cutin hydrolysate was not inhibited by actinomycin D and was stimulated ( approximately 100%) by cordycepin. Addition of cycloheximide with the inducer, or up to 12 h after the addition of the inducer, resulted in a nearly immediate cessation of cutinase production. Deoxyglucose, an inhibitor of proten glycosylation, inhibited the induction of cutinase by cutin hydrolysate. omega-Hydroxy fatty acids were more effective in inducing cutinase than any of the other more polar acids of cutin. Experiments with derivatives and analogues of omega-hydroxy C(16) acid indicated that a free hydroxyl group at the omega-position was the most important factor determining the cutinase-inducing activity. n-Aliphatic primary alcohols with 14 or more carbon atoms induced cutinase, and n-C(16) was the most effective inducer. These results strongly suggest that the monomers function as the chemical signal which induces the extracellular hydrolase.