Differential mutagenicity of streptozotocin analogs of the carbohydrate moiety

The mutagenicity of streptozotocin (SZN), 8 of its analogs and N-msthyl-N-nitrosourea (MNU) were compared in Salmonella typhimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-1 analogs tested were α- and β-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; in 2 analogs glucose was replaced by α- or β-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into 4 groups which differed by about 10-fold in mutagenicity from one another. The most mutagenic was (i) SZN, followed by (ii) β-methyl-SZN; (iii) α-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; (iv) α and β-inositol-MNU. These results suggest that the presence of the glucose moiety is conducive to a high level of mutagenicity of SZN. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the α position lead to decreased mutagenicity. The least mutagenic analogs are those in which the glucose moiety is replaced by inositols.The mutagenicity of SZN, β-methyl-SZN and of β-butyl-SZN was also compared in a mouse tissue-mediated assay. SZN was about 500-fold more mutagenic than its β-methyl analog, while the β-butyl analog was not mutagenic.Depletion of SZN and 4 of its analogs from the medium in presence of bacteria was determined spectrophotometrically. The more mutagenic compounds were depleted more rapidly but the quantitative differences in mutagenicity between these compounds could not be accounted for by depletion alone.     

Effects of l-cysteine and O-acetyl-l-serine in the synthesis and mutagenicity of azide metabolite

The ability of L-cysteine to inhibit azide-metabolite synthesis and mutagenecity is investigated in Salmonella typhimurium TA1530 and cys E₆ strains. L-cysteine specifically inhibits the synthesis of the mutagenic azide metabolite as other compounds containing SH group did not affect the production of this metabolite. Azide mutagenicity is completely inhibited by L-cysteine at a concentration (5 μmoles/plate) where the metabolite mutagenicity was not affected. $\text{O-}\text{Acetyl-}\text{L}\text{-serine}$ can reverse the L-cysteine mediated inhibition of the metabolite synthesis and thus mutagenicity in the same strains. These results suggest that $\text{O-}\text{acetyl-}\text{L}\text{-serine}$ may be required to synthesize the azide metabolite or its precursor.     

Structure—activity relationships in the mutagenicity of quinone methides of 7-hydroxyflavylium salts for Salmonella typhimurium

Several synthetic 7-hydroxyflavylium salts related to apigeninidin, a natural 3-deoxyanthocyanidin, have been studied in the Ames mutagenicity test using strain TA1537 of Salmonella typhimurium. Under the neutral pH conditions of the test, these flavylium salts are deprotonated through ionization of the C7-OH (pK_a′ = 4.2–4.4) to form quinone methides. Only the quinone methides of 4-methyl-7-hydroxyflavylium chloride and 4′-methoxy-4-methyl-7-hydroxy-flavylium chloride showed mutagenicity. Responses of 4–8 times the background were observed at the higher doses (1000 μg/plate), both with and without metabolic activation. It was concluded that the induction of frameshift mutagenicity by this group of compounds is caused by those quinone methides that have non-ionic, stable polycyclic structures at neutral pH.     

Mutagenic selectivity of carcinogenic nitroso compounds I. N-methyl-N-nitrosourethane

The mutagenicity of the carcinogen methylnitrosourethane (MNUr) was examined in Drosophila with a view to the determination of its activity on heterochromatic loci (especially rDNA) relative to those in the euchromatin. Assays were made of the yield of rDNA mutations (bobbed: bb) relative to other X-chromosome recessive lethals and visibles [X(l + v)] in the same male germ cells after treatment with different doses (1–10 mM) and at various stages in spermatogenesis.Dose dependence followed the same pattern for all genic loci and germ cell stages. In all instances, the regression of mutation frequency on injected molar dose was approximately linear, but could better be described by a quadratic dose curve. In contrast, the mutagenicity pattern during spermatogenesis varied according to the target genes. The response of the euchromatic loci reached a peak among the earlier germ cells (probably the spermatocytes), whereas that for the heterochromatic sites (including rDNA) was maximal in mature sperm.Mutagenic selectivity for rDNA with MNUr, as indicated by the percentage bb/X-mutations, was among the highest for the intrinsically reactive carcinogens (alkylating and arylating agents). This correlates with the strong carcinogenicity of MNUr and adds further support to the concept that rDNA mutations might well be a crucial step in cancer initiation.     

Mutagenicity of dimethylnitrosamine in the metabolic process by rat liver microsomes

The mutagenicity of dimethylnitrosamine (DMN) for bacteria was investigated by means of the metabolic activation process of the compound with rat liver microsomes.Three strains of streptomycin (SM)-dependent Escherichia coli having tetracycline (TC)-resistance factor (Sd-E. coli(TC)) were derived for this study. The reverse mutation in these strains from SM dependence to non-dependence was used as the marker for mutagenicity. The drug resistance factor (R factor) which was transferred to these strains was used in order to get around the bacterial contamination throughout the experiments. The study of the mutagenicity of DMN metabolites has been made by incubating DMN with rat liver microsomes and cofactor system in the presence of indicator bacterial cells.The reverse mutation was markedly induced for all of three strains in the complete incubation mixture but it was not observed when the cofactor system was omitted or the liver microsomal suspension was replaced by the kidney cell sap. When the indicator bacterial cells were added to the mixture in which DMN was previously incubated with the microsomes and cofactor system, the mutagenicity was extremely decreased.     

Mutagenicity of benzidine and 4-aminobiphenyl after metabolic activation with isolated hepatocytes and liver 9000 × g supernatant from rat,hamster and guinea pig

The mutagenicity of benzidine and 4-aminobiphenyl towards Salmonella typhimurium strain TA1538 was measured in the presence of isolated hepatocytes from rat, hamster and guinea pig. The mutagenic potency of these compounds was also assayed with S9 (9000 × g supernatant) prepared from disrupted hepatocytes of these aryl amines was investigated.For all 3 animal species it was found that the mutagenicity of benzidine is higher with intact hepatocytes than with S9 prepared from disrupted hepatocytes. Addition of acetyl coenzyme A to the S9 fraction increased the mutagenicity of benzidine. In contrast to benzidine, the mutagenicity of 4-aminobiphenyl appeared to be lower with hepatocytes than with S9. Addition of acetyl coenzyme A to the S9 fraction decreased the mutagenicity of 4-aminobiphenyl.The mutagenic potency of 4-aminobiphenyl was almost equal in the presence of the liver preparations from the 3 different species, whereas obvious species differences were seen with benzidine.     

Analysis of photosynthetic pigments and chlorophyll fluorescence characteristics of different strains of Porphyra yezoensis

The levels of photosynthetic pigments and chlorophyll fluorescence of Porphyra yezoensis strains selected from high-light environments were investigated. Sutong and Sulian strains originated from the same high-light environment but were selected from different sites on the Yellow Sea coast of Jiangsu Province, China. In January (a low temperature period), the Sulian strain and the WT (a widely cultivated strain) had higher levels of chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin, and higher actual photochemical efficiency of PSII (ΔF/F _m′) than the Sutong strain. This indicated that Sulian and the WT may have better adaptation to low temperature. In March (an optimal temperature period), Sutong had higher levels of photosynthetic pigments and higher ΔF/F _m′ than the WT and Sulian strains. This suggested that Sutong had higher light use efficiency at optimal temperatures and that most energy absorbed by PSII was used for photosynthetic electron transport. The differing areas of origin of these strains may have resulted in these differences in temperature adaptation.     

The dosing determines mutagenicity of hydrophobic compounds in the Ames II assay with metabolic transformation: Passive dosing versus solvent spiking

The Ames II bacterial mutagenicity assay is a new version of the standard Ames test for screening chemicals for genotoxic activity. However, the use of plastic micro-titer plates has drawbacks in the case of testing hydrophobic mutagens, since sorptive and other losses make it difficult to control and define the exposure concentrations, and they reduce availability for bacterial uptake or to the S9 enzymes. With passive dosing, a biocompatible polymer such as silicone is loaded with the test compound and acts as a partitioning source. It compensates for any losses and results in stable freely dissolved concentrations. Passive dosing using silicone O-rings was applied in the Ames II assay to measure PAH mutagenicity in strains TA98 and TAMix – a mixture of six different bacterial strains detecting six different base-pair substitutions – after metabolic activation by S9. Initially, 10 PAHs were tested with passive dosing from saturated O-rings, aiming at levels in the test medium close to aqueous solubility. Fluoranthene, pyrene and benzo(a)pyrene were mutagenic in both TA98 and TAMix, whereas benz(a)anthracene was mutagenic in TA98 only. The concentration-dependent mutagenic activity of benzo(a)pyrene was then compared for passive dosing and solvent spiking. With spiking, nominal concentrations greatly exceeded aqueous solubility before mutagenicity was observed, due to sorptive losses and limiting dissolution kinetics. In contrast, the passive dosing concentration-response curves were more reproducible, and shifted towards lower concentrations by several orders of magnitude. This study raises fundamental questions about how to introduce hydrophobic test substances in the Ames II assay with biotransformation, since the measured mutagenicity not only depends on the compound potency but also on its supply, sorption and consumption during the assay.     

Der respiratorische Stoffwechsel von Calliphoridenlarven in Beziehung zu Temperaturadaptation und Regulation

The oxygen consumption of five larval stages of the Calliphoridae Callitroga macellaria, Lucilia cuprina, and Calliphora vicina was analysed at different temperatures. There are differences in temperature adaptation in these three species as well as in the feeding and non-feeding larvae of Callitroga.Within fixed limits of tolerance the reaction to changes in temperature is more intensive in feeding than in non-feeding larvae. A high metabolic rate is related to high levels of sensitivity to temperature changes.At particular temperatures oxygen consumption generally decreases with age. The results partly confirm the well-established fact that at the same temperature the metabolic rate of ‘cold’-adapted stages and species is relatively high as compared to ‘warm’-adapted ones.During the course of experiments oxygen consumption was frequently inconstant as can be seen by comparing the oxygen consumption of the first and second parts of the experiments, or more exactly, by regression analysis of the oxygen consumption rates throughout these experiments. Increasing and decreasing oxygen consumption depends on temperature adaptation which indicates regulatory processes.A method to determine standard temperature using measurements of respiratory metabolism is discussed.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Potential cytotoxic and mutagenic effect of Pinus wallichiana,Daphne oleiodes and Bidens chinensis

The Pinus wallichiana, Daphne oleiodes and Bidens chinensis have a long history of being used traditionally for treatment of various types of disorders. Most of the uses have been without any scientific evidence and toxicological assessment. We evaluated the mutagenic and cytotoxic capabilities of various parts of P. wallichiana, D. oleoides and B. chinensis. Ames Salmonella mutagenicity assay determined the mutagenicity activity against TA 98 and TA 100 bacterial strains of Salmonella typhimurium without metabolic activator S9 system. The number of mutant colonies in negative control was considered as limit to determine the mutagenicity effects of every extract. Brine shrimps lethality bioassay was used to determine the cytotoxic capabilities of the selected plants. The P. wallichiana, D. oleiodes and B. chinensis did not showed any mutagenic activity both for frameshift mutation (TA98) and base-pair substitution (TA100) without S9 mixture. The crude methanolic extract of P. wallichiana stem showed moderate cytotoxicity (53.33%) at 1000 μg/ml with LD₅₀ value 599.634. The D. oleoides fruit showed a toxicity of 60% at 1000 μg/ml with LD₅₀ value 367.730. The B. chinensis (whole plant) showed lethality of 63.3% at 1000 μg/ml, with LD₅₀ 204.833. The absence of any mutagenic activity of crude extract of the tested plants in both bacteria strains, TA 98 and TA 100 without the S9 mix confirms the safety of these plants to the consumers.     

Mutagenicity assessment of Salacia chinensis by bacterial reverse mutation assay using histidine dependent Salmonella typhimurium tester strains

Background and objectiveGenotoxicity analysis is one of the most important non-clinical environmental safety investigations required for pharmaceutical and agrochemical product registration. Any medicinal product must undergo a risk evaluation to determine its mutagenicity and carcinogenicity.Materials and methodsThe Ames test is a commonly used in vitro test for determining a test chemical's mutagenic activity. Histidine-dependent Salmonella typhimurium strains with a defective gene that causes the bacteria to synthesis the necessary amino acid histidine for life were tested for mutagenic potential. In order to reveal pro-mutagens and mutagens, the mutagenic potential of both plate integration and pre-incubation techniques was examined in the presence and absence of metabolizing system. Salacia chinensis has been widely used in ayurveda to treat various ailments. However, the information of mutagenicity of Salacia chinensis is scarce as per available literature.ResultsThe mutagenicity of a Salacia chinensis root extract was investigated utilizing the Ames assay with plate incorporation and pre-incubation protocols using the appropriate Salmonella typhimurium tester strains: TA98, TA100, TA1537, TA1535, and TA102 in the presence and absence of S9. The concentrations used were 0.3123, 0.625, 1.25, 2.5 and 5 mg/plate. The extract of Salacia chinensis root did not show any mutagenic effect in any of the Salmonella typhimurium strains at the concentrations tested in the absence or presence of metabolic activation.ConclusionThe root of Salacia chinensis was hence confirmed to be non-mutagenic and at least according to the results of this genotoxicity evaluation can be regarded as being safe for human use.     

Mutagenic effect of furocoumarin monoadducts and cross-links on bacteriophage lambda

The mutagenicities of 17 closely related oxiranes were determined in 4 tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537). The test compounds comprised all possible oxides of benzene and its partially hydrogenated congeners. In TA100 and TA1535, 12 of the tested oxiranes were weak to moderate mutagens. 4 of these were also active in TA98. No mutagenicity was observed with the remaining 5 compounds in any of the 4 strains.The presence of a double bond in formal conjugation with the epoxide ring increased the mutagenicity relative to that of the saturated oxirane. Interestingly, additional epoxide rings within the same molecule did not markedly increase the mutagenic activity, and for the oxiranes that are not activated by a double bond, the relationship between mutagenic activity and the number of epoxide rings in the molecule was even inverse.The influence of bromo and hydroxyl substitution on oxirane mutagenicity is discussed. Most notably, a compound having a 4-hydroxyl group in syn position to a 1,2-epoxide ring fused to the cyclohexane ring, a structure which has been suggested to increase the electrophilic reactivity of dihydrodiol epoxides through hydrogen bonding, was almost inactive.     

Mutagenic selectivity of carcinogenic nitroso compounds: II. N,N-Dimethylnitrosamine

The genetic properties of the hepatocarcinogen N,N-dimethylnitrosamine (DMN) were examined in Drosophila for the assessment of the role of dose, cellular metabolism and genic target in its mutagenicity. Genetic activity was assayed with respect to the induction of the non-specific X-chromosome recessives (lethals and visibles) relative to the effects on specific genic sites, especially rDNA, which yields bobbed (bb) mutations.Dose dependence followed a quadratic course for all mutational classes and germ cell types, which indicated that DMN induced at least some multiple-hit mutagenic events. The genetic activity of DMN was favoured by cellular metabolism for all mutational classes, as was indicated by the progressive increase in mutation yield during spermatogenesis — from the metabolically inert mature sperm to the actively metabolizing spermatocytes and spermatogonia.The role of DNA methylation in the mutagenicity of DMN was deduced from quantitative assays of its genetic activity relative to the methylating nitrosamide — N-methyl-N-nitrosourethane (MNUr) — over the same dose range (1–10 mM) and on identical cell types and genic targets. In the metabolically inert cells (mature sperm), the two compounds were equally active with respect to the non-specific effects (X-recessives), but MNUr was considerably more effective on rDNA (bb's). Conversely, in the metabolically active cells (spermatocytes and spermatogonia), DMN exerted a much higher non-specific mutagenicity than MNUr, but the two compounds were equally effective on rDNA. These results could not be entirely interpreted by the methylation hypothesis and indicated that a DMN aldehydic metabolite, structurally analogous to MNUr, might be responsible for the induction of the rDNA mutations.The rDNA selectivity index of DMN was significantly lower than for MNUr, which paralleled their relative carcinogenic versatilities. However, DMN was comparatively more effective on the tRNA genes, a feature which might be associated with its oncogenic specificity.     

Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA 1538

A series of ten azo dyes as well as various single ring aromatic amines substituted on the benzene ring were tested for bacterial mutagenicity with Salmonella typhimurium TA 1538 using a soft-agar overlay method. Two dyes, sudan 2 and chrysoidin induced mutation but only in the presence of a rat liver preparation. Chrysoidin was the more active. Testing of its reduction products, aniline and 1,2,4-triaminobenzene showed a liver metabolite of the latter compound could be responsible for the mutagenic effect, having a comparable mutagenicity with 1,2-diamino-4-nitro-benzene, one of the mutagenic constituents of hair dyes. Structure-activity studies on a series of ring-substituted anilines indicated that mutagenic activity required at least two positions to be substituted with either amino or nitro groups, or one of each. The bacteria as well as the liver enzyme preparation may partake in the activation of these chemicals. The correlation between mutagenicity and carcinogenicity for this group of compounds is discussed.     

Effect of temperature and salinity on the energetics of juvenile gray snapper (Lutjanus griseus): implications for nursery habitat value

Gray snapper (Lutjanus griseus) encounter a wide range of temperatures and salinities in nearshore and estuarine juvenile habitats. The energetic response of juvenile gray snapper to temperature and salinity was measured in laboratory experiments to determine the influence of these physicochemical factors on the potential value of different juvenile nurseries. Maximum consumption and growth rates of juvenile (25-50 mm SL) gray snapper were determined in 12-day trials at 20 temperature/salinity combinations representing conditions in juvenile habitats. Ad libitum feeding level of individual fish was measured daily. Maximum weight specific feeding rate increased significantly with temperature and salinity; however, the effect of salinity was much less than that of temperature. Linear growth rate and specific growth rate both increased with temperature, and salinity did not have a significant effect on either. Gross growth efficiency (K₁, growth×consumption⁻¹*100) increased with temperature and was significantly lower at high salinities, indicating increased energetic costs. The higher K₁ at lower salinities has several implications for juvenile gray snapper in low salinity habitats: (1) they would need less food to achieve the same somatic growth as juveniles in high salinity habitats; (2) they would have higher growth at limited ration levels as compared to high salinity habitats; and (3) they would have less impact on prey populations than higher salinity habitats assuming similar gray snapper densities.     

Production of Pectate Lyases and Cellulases by Chryseomonas luteola Strain MFCL0 Depends on the Growth Temperature and the Nature of the Culture Medium: Evidence for Two Critical Temperatures

Several extracellular enzymes that are responsible for plant tissue maceration were detected in culture supernatant of the psychrotrophic bacterium Chryseomonas luteola MFCL0. Isoelectrofocusing experiments showed that pectate lyase (PL) activity resulted from the cumulative action of three major isoenzymes, designated PLI, PLII, and PLIII. Cellulolytic activity was also detected in culture supernatants. These enzymes exhibited different behaviors with respect to growth temperature. PLII was not regulated by temperature, whereas PLI and PLIII were regulated similarly by growth temperature. Maximal levels of PLI and PLIII were produced at 14°C when cells were grown in polygalacturonate-containing synthetic medium and at around 20 to 24°C in nutrient broth. In contrast, thermoregulation of cellulolytic activity production differed from thermoregulation of PL. The level of cellulolytic activity was low in all media at temperatures up to 20°C, and then it increased dramatically until the temperature was 28°C, which is the optimal temperature for growth of C. luteola. Previously, we defined the critical temperature by using the modified Arrhenius equation to characterize bacterial behavior. This approach consists of monitoring changes in the maximal specific growth rate as a function of temperature. Our most striking result was the finding that the temperature at which maximum levels of PLI and PLIII were produced in two different media was the same as the critical temperature for growth observed in these two media.     

Glacial clay affects foraging performance in a Patagonian fish and cladoceran

Climate change is altering temperatures and precipitation patterns all over the world. In Patagonia, Argentina, predicted increase in precipitation together with rapidly melting glaciers increase the surface runoff, and thereby the transport of suspended solids to recipient lakes. Suspended solids affect the visual conditions in the water which in turn restricts visual foraging. The native fish Aplochiton zebra Jenyns, and its filter-feeding cladoceran prey, Daphnia commutata Ekman, were subjected to foraging experiments at three turbidity levels. A. zebra foraging rate was substantially reduced at naturally occurring turbidity levels and the filtering rate of D. commutata was reduced at the highest turbidity level. This indicates that Daphnia may be partly released from predation from A. zebra at the same time as it can maintain relatively high feeding rates as turbidity increases. Lower foraging rates at the same time as the metabolic demand increases, through increased temperatures, may result in larger effects on A. zebra than could be expected from increases in turbidity or temperature alone. Turbidity may, as an indirect effect of climate change, decrease planktivore foraging rates and thereby alter the interaction strength between trophic levels.