Gas Chromatography/Mass Spectrometry-Based Metabolomic Profiling Reveals Alterations in Mouse Plasma and Liver in Response to Fava Beans

Favism is a life-threatening hemolytic anemia resulting from the intake of fava beans by susceptible individuals with low erythrocytic glucose 6-phosphate dehydrogenase (G6PD) activity. However, little is known about the metabolomic changes in plasma and liver after the intake of fava beans in G6PD normal and deficient states. In this study, gas chromatography/mass spectrometry was used to analyze the plasma and liver metabolic alterations underlying the effects of fava beans in C3H- and G6PD-deficient (G6PDx) mice, and to find potential biomarkers and metabolic changes associated with favism. Our results showed that fava beans induced oxidative stress in both C3H and G6PDx mice. Significantly, metabolomic differences were observed in plasma and liver between the control and fava bean treated groups of both C3H and G6PDx mice. The levels of 7 and 21 metabolites in plasma showed significant differences between C3H-control (C3H-C)- and C3H fava beans-treated (C3H-FB) mice, and G6PDx-control (G6PDx-C)- and G6PDx fava beans-treated (G6PDx-FB) mice, respectively. Similarly, the levels of 7 and 25 metabolites in the liver showed significant differences between C3H and C3H-FB, and G6PDx and G6PDx-FB, respectively. The levels of oleic acid, linoleic acid, and creatinine were significantly increased in the plasma of both C3H-FB and G6PDx-FB mice. In the liver, more metabolic alterations were observed in G6PDx-FB mice than in C3H-FB mice, and were involved in a sugar, fatty acids, amino acids, cholesterol biosynthesis, the urea cycle, and the nucleotide metabolic pathway. These findings suggest that oleic acid, linoleic acid, and creatinine may be potential biomarkers of the response to fava beans in C3H and G6PDx mice and therefore that oleic acid and linoleic acid may be involved in oxidative stress induced by fava beans. This study demonstrates that G6PD activity in mice can affect their metabolic pathways in response to fava beans.     

Pathophysiology of favism

Haemolytic crises occurring in G6PD-deficient individuals after ingestion of fava beans (favism) are much less frequent than in the past. However, favism is a unique natural model of oxidant damage in vivo, useful for the study of senescent or damaged red blood cells (RBC) clearance from circulation. The following aspects have been considered: 1. Pathophysiology of favism, including incidence, salient features, and sequence of events. 2. RBC alterations during the haemolytic crisis: biochemical, rheological and morphological alterations occurring in RBC isolated at different stages of the crisis. 3. Toxic substances of Vicia faba and their mechanism of action: treatment of G6PD-deficient RBC with divicine or isouramil (redox substances present in fava beans) provokes the same changes as observed during favism. 4. Intravascular vs. extravascular haemolysis: extravascular (i.e. phagocytic) removal of damages RBC seems predominant in favism. 5. The signal for RBC removal: in analogy with a recent model for recognition and removal of oxidant-stressed or senescent RBC, we propose removal of fava bean damaged RBC be mediated by apposition of antiband 3 antibodies and complement C3 fragments, recognized as non-self recognition signal by monocytes and macrophages.     

Glucose-6-phosphate dehydrogenase deficiency among the student population of Milan

In our Department a yearly screening is carried out on the school population of Milan, in order to detect beta-thalassaemic trait carriers and G-6-P-D deficient males. This report deals with the results obtained on a group of 21,118 males. They all showed very low enzyme activity and often just detectable; the fall of GSH after incubation with acetylphenhylhidrazine appeared significantly greater than normal. Only 3 cases of hemolytic crises by fava beans ingestion and 7 cases of neonatal jaundice resulted from anamnestic data.     

The mutagenic activity of gastric juice

The mutagenic activity of fasting gastric juice was assessed in 123 patients including 18 with normal endoscopic findings, 53 peptic ulceration, 9 gastric cancer, 12 pernicious anaemia and 31 patients who had undergone peptic ulcer surgery in the past. Significant mutagenic activity was detected in 96 (78%). Marked variations in mutagenic activity were noted both within and between the patient groups and no significant differences were detected. No correlation was found between mutagenic activity and patient age or sex, gastric pH, bile acid concentrations or bacterial counts, intestinal metaplasia on gastric mucosal biopsy, or intragastric nitrite. About 30% of gastric juice samples showed evidence of a cytotoxic activity towards the Salmonella tester strains in the mutation assay. Preliminary studies on other body fluids showed the presence of significant mutagenic activity in fasting saliva, bile and plasma. These findings demonstrate widespread human exposure to potentially genotoxic substances.     

Effect of histamine H2-receptor antagonist therapy on the mutagenic activity of gastric juice

There is concern at present that treatment with histamine H2-receptor antagonists might promote the development of gastric cancer by producing conditions which favour intragastric formation of N-nitroso compounds. If H2-receptor antagonist therapy causes increased intragastric levels of N-nitroso compounds, an issue not yet resolved by analytical studies, corresponding changes in the mutagenic activity of gastric juice might be anticipated. In this study mutagenic activity and pH were measured in fasting gastric aspirate from 18 peptic ulcer patients before and during the final week of therapy with ranitidine (n = 10) or cimetidine (n = 8). Mutagenic activity was assessed using Salmonella typhimurium TA98 and TA100 in a modified pre-incubation "fluctuation" test. No significant change in mutagenic activity was detected after therapy. Of 15 patients found to have significant mutagenic activity in their fasting gastric juice before treatment, 14 remained mutagenic following treatment. Mutation frequencies (sum of positive wells in duplicate 96-well microtitre plates, mean +/- SD) for TA98 and TA100 were respectively, 20 +/- 34 and 100 +/- 64 before compared with 10 +/- 6 and 102 +/- 65 after therapy (p greater than 0.05). Changes in mutagenic activity were similar in both treatment groups and unrelated to duration of therapy, changes in gastric pH or ulcer healing. In vitro, neither cimetidine in aqueous solution, nor gastric juice preincubated with cimetidine showed significant mutagenic activity. These results provide no evidence that increased intragastric levels of genotoxic chemicals, such as N-nitroso compounds, occur during H2-receptor antagonist therapy.     

Evaluation of antioxidant activity of leafy vegetables and beans with myoglobin method

Key message

Antioxidant activity of seven leafy vegetables and four beans against five reactive oxygen species and reactive nitrogen species was clearly characterized with a protocol using myoglobin as a reporter probe.

Abstract

Antioxidant activity of seven leafy vegetables and four beans against peroxyl radical, hydroxyl radical, hypochlorite ion, and peroxynitrite ion has been measured using myoglobin as a reporter probe (myoglobin method). Conventional DPPH method was also used to evaluate antioxidant activity of the samples. Difference of activity against different reactive oxygen species (ROS) and reactive nitrogen species (RNS) was characterized by plotting the data in a 5-axe cobweb chart. This plot clearly showed the characteristics of the antioxidant activity of the leafy vegetables and the beans. The samples examined in this work were categorized into four groups. (1) The samples showed high antioxidant activity against all ROS and RNS: daikon sprout, spinach, Qing-geng-cai, and onion. (2) The samples showed high antioxidant activity against peroxyl radical: red bean and soy bean. (3) The samples showed high antioxidant against hypochlorite ion: broccoli floret, cabbage, and Chinese cabbage. (4) The samples showed weak antioxidant activity against all ROS and RNS: cowpea and common beans. Our protocol is probably useful to characterize antioxidant activity of the crops of different cultivars, the crops obtained in different growing environments and growing seasons, the crops harvested at different age, and the crops stored in the different conditions, as well as the changes of activity during cooking process of the crops.     

Genotoxicity of heated cooking oil vapors.

Epidemiological studies of lung cancer in Chinese women indicated that factors other than cigarette smoking are related to lung cancer risk. A case-control study suggested that indoor air pollution, particularly from cooking oil emissions, may be involved. Condensates of volatile emissions from rapeseed and soybean cooking oils were prepared and found to be genotoxic in short-term tests including the Salmonella mutation assay, SV50 forward-mutation assay, and sister-chromatid exchange assay, as well as the micronucleus assay in mouse bone marrow. In contrast, condensates from rapeseed oil with butylated hydroxyanisole or hydrogenated rapeseed oil were not mutagenic, implicating oxidation products as the cause for mutagenicity. Peanut oil and lard condensates were not mutagenic in any assay. The association of exposure to Chinese rapeseed cooking-oil emissions and lung-cancer risk may be related to the mutagenic component of these condensates.     

High mutagenic activity formed in pan-broiled pork

Lean pork was pan-broiled at various temperatures between 100 and 290 degrees C. Cooking was performed in an open frying pan common for domestic use in Sweden. No fat was added. Cooking procedures are clearly defined in order to facilitate inter-laboratory comparisons. The crust was extracted with organic solvents of varying polarity. The mutagenic activity was assayed with Ames' Salmonella mutagenicity test. Large amounts of mutagenic activity were detected in samples pan-broiled at 200-290 degrees C. The mutagenic activity recovered was about 10 times higher than that reported by previous investigators to be found during cooking of meat under similar conditions. This discrepancy could be due to differences in the composition of Swedish pork as compared to the meat samples used by other investigators or to different methodology in cooking and extraction procedures.     

Nitrate reductase and nitrogenase activities of common beans (Phaseolus vulgaris L.) from different geographic locations

Summary Cultivars ofPhaseolus vulgaris (L.) from contrasting geographic locations were cultivated under fields conditions for measurements of nitrogenase and nitrate reductase activities. A first trial with two cultivars indicated that a tropical cultivar B-789 has a higher nitrogenase activity than a temperate one Elsa. And the converse was true for the nitrate reductase activity. While where a post flowering application was made, a renewal of nitrate reductase activity occurred. Further similar comparisons of both enzymatic activities upon eight tropical and temperate cultivars of equivalent vegetative cycles indicated, on the average, that tropical cultivars have a higher level of (C₂H₂) reduction and a lower nitrate reductase activity than temperature cultivars. These observations suggest that there exists an inverse relationship between the two enzymatic activities in common beans, and there probably exists genetic variability for a possible improvement of N-fixation ability. An early application of N-fertiliser upon the Elsa and B-789 plots promotes later nitrogenase activity while a post flowering application shows obvious a renewal of nitrate reductase activity. Thus, analyses of nitrate reductase and nitrogenase activities of a common bean crop at different physiological stages may give us an indication of the best time to apply supplementary nitrogen fertilisation to common beans to increase seed yield.     

Urinary 1-hydroxypyrene and mutagenicity in bus drivers and mail carriers exposed to urban air pollution in Denmark

BACKGROUND: Previous studies in Denmark have shown that bus drivers and tramway employees were at an increased risk for developing several types of cancer and that bus drives from central Copenhagen have high levels of biomarkers of DNA damage.AIMS: The present study evaluates 1-hydroxypyrene concentrations and mutagenic activity in urine as biomarkers of exposure in non-smoking bus drivers in city and rural areas on a work day and a day off and in non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). METHODS: Twenty-four hour urine samples were collected on a working day and a day off from 60 non-smoking bus drivers in city and rural areas and from 88 non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). The concentration of 1-hydroxypyrene was measured by means of HPLC and the mutagenic activity was assessed by the Ames assay with Salmonella tester strain YG1021 and S9 mix. The N-acetyltransferase (NAT2) phenotype was used as a biomarker for susceptibility to mutagenic/carcinogenic compounds. RESULTS: Bus drivers excreted more 1-hydroxypyrene in urine than did mail carriers. The differences were slightly smaller when NAT2 phenotype, cooking at home, exposure to vehicle exhaust, and performing physical exercise after work were included. The NAT2 slow acetylators had 29% (1.29 [CI: 1.15-1.98]) higher 1-hydroxypyrene concentrations in urine than the fast acetylators. Male bus drivers had 0.92 revertants/mol creatinine [CI: 0.37-1.47] and female bus drivers 1.90 revertants/mol creatinine [CI: 1.01-2.79] higher mutagenic activity in urine than mail carriers. CONCLUSION: The present study indicates that bus drivers are more exposed to polycyclic aromatic hydrocarbons (PAH) and mutagens than mail carriers. Mail carriers who worked outdoors had higher urinary concentration of 1-hydroxypyrene, a marker of exposure to PAH, than those working indoors. The individual levels of urinary mutagenic activity were not correlated to excretion of 1-hydroxypyrene. This might be due to the fact that the most potent mutagenic compounds in diesel exhaust are not PAH but dinitro-pyrenes. Among bus drivers, fast NAT2 acetylators had higher mutagenic activity in urine than slow NAT2 acetylators and female bus drivers had higher mutagenic activity than male bus drivers.     

The effect of temperature on the formation of mutagens in heated beef stock and cooked ground beef

The microsome-activatable mutagens (chromatographically distinguishable from benzo[a]pyrene and from the mutagens produced from pyrolysed amino acids and proteins) previously found in beef extract and in bacterial nutrients which contain beef extract are produced when beef stock is heated. Reflux boiling of beef stock at 100°C results in a linear increase in mutagenic activity toward Salmonella strain TA1538. The rate of production of mutagenic activity at temperatures between 68°C and 98°C conforms closely to the Arrhenius equation, yielding an activation energy of 23 738 calories per mole. Extrapolation from these data predicts a sharp rise in the rate of mutagen formation between 140 and 180°C. This expectation is confirmed when ground beef patties (hamburgers) are prepared in various conventional electrically-heated appliances which operate at different cooking temperatures within this range. The mutagenic activity of hamburger cooked at high temperatures is limited to the surface layers; the temperature of the inside of the hamburger does not exceed 100°C during cooking. No mutagenic activity is found in comparable samples of uncooked meat. The results indicated that the mutagens may be formed as a result of the temperatures encountered in certain conventional cooking procedures.     

Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

Compatibility of Rhizobial Genotypes within Natural Populations of Rhizobium leguminosarum Biovar viciae for Nodulation of Host Legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

Microwave-induced stimulation of l-DOPA,phenolics and antioxidant activity in fava bean (Vicia faba) for Parkinson’s diet

Fava bean sprouts are rich in levo-dihydroxy phenylalanine (l-DOPA), the precursor of dopamine, and are being investigated for use in the management of Parkinson’s disease. The phytopharmaceutical value was improved during germination by a microwave treatment of the seeds, the phenolic content of the germinated sprouts increasing 700% and l-DOPA content by 59% compared to control. A higher antioxidant activity that was observed correlated with total phenolics and l-DOPA contents. The glucose-6-phosphate dehydrogenase activity peaked on the seventh day of germination with a concurrent increase in phenolics indicating enhanced mobilization of carbohydrates. A higher guaiacol peroxidase activity was observed indicating an increased polymerization of phenolics. The elevated superoxide dismutase activity was proportional to the stimulation of antioxidant activity. The major implication from this study is that microwave treatment can significantly stimulate the phenolic antioxidant activity and Parkinson’s relevant l-DOPA content of fava beans sprouts.     

Mutagenic properties of spent bleaching liquors from sulphite pulps and a comparison with kraft pulp bleaching liquors

Parameters influencing the mutagenic properties of spent bleaching liquors from sulphite pulps have been studied. In addition a comparison has been made between the properties of spent liquors from sulphite and kraft pulp bleaching. In the sulphite process the cooking base had no influence on the mutagenicity of the chlorination stage. In contrast, removing the extractives before chlorination especially for dissolving pulp resulted in an increase in mutagenic activity. The mutagenicity decreased significantly after substituting 40% of the chlorine with chlorine dioxide. Sequential addition of chlorine and chlorine dioxide resulted in higher activity than simultaneous or premixed chlorination as observed for liquors from kraft pulp. Increasing the pH of the extracts or addition of sulphur dioxide decreased the mutagenicity. Expressed as 10(7) revertants per kappa number and ton pulp the mutagenicity varied between 10 and 40 for sulphite pulp while the corresponding figures for kraft pulp were 100-225.     

Genetic diversity and phylogeny of Japanese sake-brewing rice as revealed by AFLP and nuclear and chloroplast SSR markers

Japanese rice (Oryza sativa L.) cultivars that are strictly used for the brewing of sake (Japanese rice wine) represent a unique and traditional group. These cultivars are characterized by common traits such as large grain size with low protein content and a large, central white-core structure. To understand the genetic diversity and phylogenetic characteristics of sake-brewing rice, we performed amplified fragment length polymorphism and simple sequence repeat analyses, using 95 cultivars of local and modern sake-brewing rice together with 76 cultivars of local and modern cooking rice. Our analysis of both nuclear and chloroplast genome polymorphisms showed that the genetic diversity in sake-brewing rice cultivars was much smaller than the diversity found in cooking rice cultivars. Interestingly, the genetic diversity within the modern sake-brewing cultivars was about twofold higher than the diversity within the local sake-brewing cultivars, which was in contrast to the cooking cultivars. This is most likely due to introgression of the modern cooking cultivars into the modern sake-brewing cultivars through breeding practices. Cluster analysis and chloroplast haplotype analysis suggested that the local sake-brewing cultivars originated monophyletically in the western regions of Japan. Analysis of variance tests showed that several markers were significantly associated with sake-brewing traits, particularly with the large white-core structure.     

Effect of tannin content of faba bean (Vicia faba) seed on seed vigour, germination and field emergence

Differences in seed vigour of zero- and high-tannin faba beans were investigated using 25 seed lots of 12 cultivars following earlier reports of poor emergence in the zero-tannin types. Field emergence ranged from 54–96% indicating differences in seed vigour between cultivars all having high laboratory germination (>91%). Seed from zero-tannin accessions with poor emergence had a higher incidence of testa and cotyledon cracking, a smaller percentage of hard seeds, more rapid water uptake, a lower percentage of vital staining of cotyledons and a greater leaching of solutes than high-tannin types. Nevertheless, variation in these characteristics existed between cultivars and lines of both types. Seeds with more cracks in the seed coat and fewer hard seeds imbibed water more rapidly and consequently showed lower levels of vital staining and more cracks in the abaxial surface of the cotyledons. Slower imbibition in polyethylene glycol lessened the incidence of these deleterious characteristics and may provide a practical resolution to the problem of poor field emergence in zero-tannin lines of faba bean with low seed vigour.     

Deficiency in OGG1 protects against inflammation and mutagenic effects associated with H. pylori infection in mouse

BACKGROUND: Helicobacter pylori infection is associated with gastric cancer. Study with the Big Blue mouse model has reported a mutagenic effect associated with the H. pylori infection, as a result in part of oxidative DNA damage. The present work investigates the consequences of a deficiency in the OGG1 DNA glycosylase, responsible for the excision of 8-oxo guanine, on the inflammatory and genotoxic host response to the infection. MATERIALS AND METHODS: Big Blue Ogg1-/- C57BL/6 mice were orally inoculated with H. pylori strain SS1 or vehicle only, and sacrificed after 1, 3, or 6 months. The serologic response, histologic lesions, mutant frequency, and spectra of mutations were assessed in the stomach and compared to what observed in the wild-type (Wt) context. RESULTS: Inflammatory lesions induced in the gastric mucosa of H. pylori-infected mice, corresponding to a moderate gastritis, were less severe in Ogg1-/- than in Wt Big Blue mice. Analysis of antimicrobial humoral immunity exhibited a lower IgG2a serum level (Th1 response) after 6 months of infection in Ogg1-/- than in the Wt mice. In these conditions, the H. pylori-SS1 infection in the Ogg1-/- mice did not induce a mutagenic effect at the gastric epithelial cells level, either after 3 or 6 months. CONCLUSIONS: The inactivation of the OGG1 DNA glycosylase in mouse leads to less severe inflammatory lesions and abolished the mutagenic effect at the gastric epithelial cells level, induced by the H. pylori infection. These data suggest for the OGG1deficiency a protective role against inflammation and genotoxicity associated to the H. pylori infection.     

Mutagens from the cooking of food: I. Improved extraction and characterization of mutagenic fractions from cooked ground beef

Ground beef was fried at 200°C (392°F) to a well-done, non-charred state, and the extracted organic base fraction was found to be highly mutagenic in Salmonella strain TA1538 (6300 revertants/100 g equivalent, gE, fresh weight). The neutral and acidic extracts showed no mutagenic activity in any of the 5 standard strains of Salmonella. A new procedure based upon extraction and protein precipitation with acetone is described, which is simpler and more efficient than previously described methods. The organic base fraction was mutagenic only in Salmonella strains TA1537, TA1538, and TA98, all sensitive to frameshift mutations. Strains sensitive to base-substitution mutations showed no activity. Metabolic activation was an absolute requirement for mutagenesis; however cell toxicity was decreased by the presence of S9 activation mixture. After normal cooking, more than 20 times as much mutagenic material remained in the meat as was recovered in the pan grease and vapors. The results confirm that mutagens are formed under conventional frying conditions, and show that mutagen can be isolated by an improved extraction method.     

Effect of Thermal Processing and Maceration on the Antioxidant Activity of White Beans

Phenolic compounds, which naturally occur in beans, are known to have antioxidant activity, which may be partially lost during the processing of this legume. This study evaluated the effect of thermal processing and maceration on the phenolic acid and flavonoids profile and content and on the antioxidant activity of white beans. According to the results obtained from the 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method, there were no significant differences among treatment groups analysed. When was using 1,1-diphenyl-2-pycrylhydrazyl method (DPPH), beans cooked without maceration present the higher antioxidant activity, and raw beans the lower. The phenolic acids found in greater amounts were gallic acid and chlorogenic acid. Kaempferol was only detected in the soaked and cooked samples; catechin and kaempferol-3-rutinoside were found in the highest concentrations. Quercetin and kaempferol-3-glucoside were not affected by the cooking process, either with or without maceration. In general, the heat treatment increased the antioxidant activity.