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The gut microbiota helps us digest food and has been associated with both good health and risk of disease. Although compositions of the gut microbiota are highly divergent, the gut microbiota exhibits a high level of functional redundancy making it difficult to reliably link gut microbiome patterns to health and diet across individuals. Thus, there is a need to move beyond profiling of the gut microbial composition to the assessment of gut microbial metabolic activity in order to expand knowledge on the impact of the gut microbiota on health. Metabolomics has already proven its utility in identifying gut‐derived microbial metabolites, which may be mediators of diet‐induced host–microbial crosstalk important for health. These diet‐derived metabolites include, among many others, the short‐chain fatty acids originating from bacterial degradation of dietary fibres and proteins, secondary bile acids derived from primary bile acids, microbial tryptophan catabolites resulting from proteolysis, imidazole propionate produced from histidine and trimethylamine N‐oxide, a host–microbial co‐metabolite of nutrients such as phosphatidylcholine, choline and L‐carnitine, present in high‐fat foods. These co‐metabolites have been associated with beneficial and detrimental effects in the host. However, the vast majority of metabolites measured by untargeted metabolomics in human blood and excreta remain unknown and may include additional microbial molecules of importance for health. Thus, there is great potential, yet to be explored, for identifying additional diet‐derived microbial products that affect the host. Herein, we review key advances, challenges and limitations in our understanding of diet–microbiome interactions and diet‐derived microbial metabolites in relation to human health and discuss how metabolomics may shed light on inter‐individual differences in dietary responses. [ABSTRACT FROM AUTHOR]

Many people who are allergic to birch pollen are also allergic to apple fruit, due to cross- allergenicity. Since apples are the most extensively consumed fruit in Europe, it is highly relevant to develop a hypo-allergenic apple. Apples with significantly reduced levels of the allergen, Mal d 1, may allow many apple allergics to eat them without an allergic reaction. We are currently collaborating to develop a hypo-allergenic apple within the European Integrated Research Project, ISAFRUIT (www.isafruit.org). Hypo-allergenic apple plants (Malus X domestica Borkh., 'Elstar') with decreased levels of Mal d 1 mRNA were produced by RNA interference (RNAi) technology. Ten genetically modified (GM) apple lines were selected. In vitro plantlets were first transferred to a greenhouse, then grafted onto wild-type M.9 rootstock to promote the development of fruit-producing trees. Levels of Mal d 1 gene silencing were measured repeatedly by quantitative real-time PCR. Compared to leaf samples from wild-type 'Elstar', two GM lines showed modest levels of gene silencing (up to 250-fold), whereas the other eight GM lines were significantly silenced (up to 10,000-fold) in Mal d 1 gene expression. These levels of silencing were unaffected by grafting, and have been stable over more than 3 years, and throughout all developmental stages. [ABSTRACT FROM AUTHOR]

Epidemiological studies suggest that foods rich in flavonoids might reduce the risk of cardiovascular disease and cancer. The objective of the present study was to investigate the effect of green tea extract (GTE) used as a food antioxidant on markers of oxidative status after dietary depletion of flavonoids and catechins. The study was designed as a 2??3 weeks blinded human cross-over intervention study (eight smokers, eight non-smokers) with GTE corresponding to a daily intake of 18??6 mg catechins/d. The GTE was incorporated into meat patties and consumed with a strictly controlled diet otherwise low in flavonoids. GTE intervention increased plasma antioxidant capacity from 1??35 to 1??56 (P<0??02) in postprandially collected plasma, most prominently in smokers. The intervention did not significantly affect markers in fasting blood samples, including plasma or haemoglobin protein oxidation, plasma oxidation lagtime, or activities of the erythrocyte superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase. Neither were fasting plasma triacylglycerol, cholesterol, ??-tocopherol, retinol, ??-carotene, or ascorbic acid affected by intervention. Urinary 8-oxo-deoxyguanosine excretion was also unaffected. Catechins from the extract were excreted into urine with a half-life of less than 2 h in accordance with the short-term effects on plasma antioxidant capacity. Since no long-term effects of GTE were observed, the study essentially served as a fruit and vegetables depletion study. The overall effect of the 10-week period without dietary fruits and vegetables was a decrease in oxidative damage to DNA, blood proteins, and plasma lipids, concomitantly with marked changes in antioxidative defence. [ABSTRACT FROM PUBLISHER]

1. Gavage administration of the natural flavonoids tangeretin, chrysin, apigenin, naringenin, genistein and quercetin for 2 consecutive weeks to the female rat resulted in differential effects on selected phase 1 and 2 enzymes in liver, colon and heart as well as antioxidant enzymes in red blood cells (RBC). 2. Glutathione transferase (GST) activity assayed by use of the substrate 1-chloro-2,4- dinitrobenzene was significantly induced by apigenin, genistein and tangeretin in the heart but not in colon or liver. 3. In RBC chrysin, quercetin and genistein significantly decreased the activity of glutathione reductase (GR), catalase (CAT) and glutathione peroxidase (GPx), whereas superoxide dismutase (SOD) was only significantly decreased by genistein. 4. The oxidative status of the animal, measured as plasma malondialdehyde, revealed that chrysin, quercetin, genistein, and beta-naphthoflavone (BNF) significantly protected against, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-induced oxidative stress. Hepatic PhIP-DNA adduct formation was not affected by any of the administered flavonoids, whereas PhIP-DNA adduct formation in colon was slightly, but significantly, inhibited by quercetin, genistein, tangeretin and BNF. 5. The observed effects of chrysin, quercetin and genistein on antioxidant enzymes, concurrently with a protection against oxidative stress, suggest a feedback mechanism on the antioxidant enzymes triggered by the flavonoid antioxidants. 6. Despite the use of high flavonoid doses, which by far exceed the human exposure levels, the effect on drug metabolizing and antioxidant enzymes was still very minor. The role of singly administered flavonoids in the protection against cancer and heart disease is thus expected to be limited. [ABSTRACT FROM AUTHOR]

Seven men and seven women participated in a randomized crossover trial to study the effect of intake of parsley (Petroselinum crispum), containing high levels of the flavone apigenin, on the urinary excretion of flavones and on biomarkers for oxidative stress. The subjects received a strictly controlled diet low in flavones and other naturally occurring antioxidants during the 2 weeks of intervention. This basic diet was supplemented with parsley providing 3·73–4·49 mg apigenin/MJ in one of the intervention weeks. Urinary excretion of apigenin was 1·59–409·09 μg/MJ per 24 h during intervention with parsley and 0–112·27 μg/MJ per 24 h on the basic diet (P < 0·05). The fraction of apigenin intake excreted in the urine was 0·58 (se 0·16) % during parsley intervention. Erythrocyte glutathione reductase (EC 1.6.4.1; GR) and superoxide dismutase (EC 1.15.1.1; SOD) activities increased during intervention with parsley (P < 0·005) as compared with the levels on the basic diet, whereas erythrocyte catalase (EC 1.11.1.6) and glutathione peroxidase (EC 1.11.1.9) activities did not change. No significant changes were observed in plasma protein 2-adipic semialdehyde residues, a biomarker of plasma protein oxidation. In this short-term investigation, an overall decreasing trend in the activity of antioxidant enzymes was observed during the 2-week study. The decreased activity of SOD was strongly correlated at the individual level with an increased oxidative damage to plasma proteins. However, the intervention with parsley seemed, partly, to overcome this decrease and resulted in increased levels of GR and SOD. [ABSTRACT FROM PUBLISHER]

In vitamin A-replete populations, increased concentrations of serum carotenoids have been associated with a decreased risk of degenerative diseases. The mechanism of action of carotenoids in determining antioxidant activity is largely unknown. The aim of the study was to examine the effect of carotenoid supplementation and spinach intake on erythrocyte enzyme antioxidant activities, serum or plasma nonenzymatic antioxidant concentrations, and concentrations of oxidatively damaged amino acids in plasma. Subjects received for 3 wk a basic diet (n = 10), a basic diet with a carotenoid supplement (n = 12) or with a spinach product (n = 12 per group), i.e., whole-leaf, minced, liquefied or liquefied spinach plus added dietary fiber. After 3 wk of dietary intervention, changes in serum or plasma concentrations of ascorbic acid, alpha-tocopherol, FRAP (ferric reducing ability of plasma) and uric acid and erythrocyte enzyme activities were assessed, and differences among experimental groups were tested. Consumption of spinach resulted in greater (P < 0.01) erythrocyte glutathione reductase activity and lower (P < 0.05) erythrocyte catalase activity and serum alpha-tocopherol concentration compared with the control group. Consumption of the carotenoid supplement led to lower alpha-tocopherol responses (P = 0.02) compared with the basic diet only. Our data suggest that the short-term changes in erythrocyte glutathione reductase activity and serum alpha-tocopherol concentration can be attributed to an increased carotenoid (lutein and zeaxanthin) intake, but beta-carotene is unlikely to be a causative factor. Lower erythrocyte catalase activity after intervention with spinach products may be related to other constituents in spinach such as flavonoids. [ABSTRACT FROM AUTHOR]

The biotransformation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]-pyridine (PhIP) and the protein binding of PhIP and 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) was studied using microsomes from PCB-pretreated or untreated male rats of the strains, Wistar, Fischer and Sprague-Dawley. The microsomal monooxygenases, P450IA1 and IA2, which are important for the biotransformation of heterocyclic amines, were quantified by immunoblots. The two metabolites detected, 2-amino-1-methyl-6-(4′-hydroxyphenyl)imidazo[4,5- b]pyridine (4′OH-PhIP) and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5- b]pyridine ( N2-OH-PhIP) were formed in similar amounts whereas no minor metabolites were found in our highly sensitive radiochemical assay. Irrespective of the rat strain used, pretreatment with PCB significantly induced both the activation and the detoxication in all three rat strains. Except for a significantly higher concentration of P450IA2 in microsomes from control and PCB induced Wistar rats, no major differences between the strains were found. [ABSTRACT FROM AUTHOR]

1. Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes. Naringenin, hesperetin, chrysin, apigenin, tangeretin, kaempferol, galangin and tamarixetin were all metabolized extensively by induced rat liver microsomes but only to a minor extent by uninduced microsomes. No metabolites were detected from eriodictyol, taxifolin, luteolin, quercetin, myricetin, fisetin, morin or isorhamnetin. 2. The identity of the metabolites was elucidated using lc-ms and H-nmr, and was consistent with a general metabolic pathway leading to the corresponding 3 ,4-dihydroxylated flavonoids either by hydroxylation or demethylation. Structural requirements for microsomal hydroxylation appeared to be a single or no hydroxy group on the B-ring of the flavan nucleus. The presence of two or more hydroxy groups on the B-ring seemed to prevent further hydroxylation. The results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C , and not at the C-position. 3. The CYP1A isozymes were found to be the main enzymes involved in flavonoid hydroxylation, whereas other cytochrome P450 isozymes seem to be involved in flavonoid demethylation. [ABSTRACT FROM AUTHOR]

We have shown previously that a high sucrose intake increases the background level of somatic mutations and the level of bulky DNA adducts in the colon epithelium of rats. The mechanism may involve either glucose or fructose formed by hydrolysis of sucrose. Male Big Blue rats were fed 30% sucrose, glucose, fructose or potato starch as part of the diet. Mutation rates and bulky DNA adduct levels were determined in colon and liver. The concentration of short-chain fatty acids and pH were determined in caecum, C-peptide was determined in plasma, biomarkers for oxidative damage and proliferation were determined in colon, and a metabonomic analysis was performed in plasma and urine. The sugars increased the mutation rates in colon and the bulky adduct levels in colon and liver to a similar extent. All sugars decrease the caecal concentration of acetic acid and propionic acid. The metabonomic studies indicated disturbed amino acid metabolism and decrease in plasma and urinary acetate as a common feature for all sugars and confirmed triglyceridemic effects of fructose. In conclusion, the genotoxicity may be related to the altered chemical environment in the caecum and thereby also in the colon but we found no related changes in insulin resistance or oxidative stress.

The influence of black currant juice, Bowman-Birk protease inhibitor (BBI), kolaviron (a biflavonoid fraction of Garcinia kola seed), sugars, vitamin C and tert-butyl hydroperoxide on a wide range of biomarkers for oxidative stress, DNA damage and sugar or lipid metabolism has been investigated in male F 344 rats. The selected pro-oxidant control, tert-butyl hydroperoxide, significantly increased plasma and liver 2-amino-adipic semialdehyde (AAS), a marker of protein oxidation (p <0.05) whereas lipid oxidation assessed as malon dialdehyde (MDA) and DNA oxidation were not significantly increased. Feeding BBI also increased the level of oxidized protein in plasma and liver at the higher dose level (0.5%). No effect was observed at the lower dose level (0.25%), which even decreased lipid oxidation in plasma. BBI did not affect background levels of DNA strand breaks or oxidation (comets). In rats exposed to black currant juice, a statistically significant decrease in liver AAS and MDA was observed. This effect could not be explained by its content of sugars or of the known redox active constituent, vitamin C. The lowering effect of black currant juice on protein and lipid oxidation was similar in magnitude to that of the known liver protectant, kolaviron. In rats treated with kolaviron (200 mg/kg body weight), background AAS levels were significantly reduced in both plasma and liver whereas the effect on MDA only reached statistical significance in plasma. Kolaviron was the only extract tested which decreased oxidative damage to DNA in the liver. The erythrocyte antioxidant enzyme activities, catalase and glutathione peroxidase were decreased in rats treated with tert-butyl hydroperoxide (p <0.05) but were not affected by the other treatments. Black currant juice and sugars increased plasma triglyceride levels and black currant juice increased plasma cholesterol but neither of them nor any other treatment affected blood glucose, erythrocyte HbA1c or fructosamine. We conclude that markers of oxidative stress may be modified by several mechanisms after feeding rats with complex dietary factors and that both pro- and antioxidant effects may consequently be observed simultaneously after short-term feeding of antioxidant-rich foods, herb medicines, or known pro- and antioxidants.

Epidemiological studies indicate that moderate alcohol consumption, particularly wine, reduce the risk of CHD. The present study was designed to investigate the effect of grape-skin extract on markers of oxidative status. The study was designed as a randomised crossover. A diet with a low content of flavonoids was served with strict control of intake in two consecutive 1-week intervention periods to fifteen subjects (nine women, six men) divided randomly into two groups. During one of the weeks the subjects from either group consumed 200 ml grape-skin extract in water (1 mg extract/ml) at each of three daily meals (31.3 mg total phenolics, including 9.0 mg catechin). An increased activity of glutathione reductase and a borderline increase of glutathione peroxidase activity in erythrocytes were observed after grape-skin intervention, while the intervention had no significant effect on superoxide dismutase or catalase. Likewise, no effect was found on 2-aminoadipic semialdehyde (AAS) residues, a plasma protein oxidation product, or on malondialdehyde in plasma or in LDL, which are markers of lipoprotein oxidation. A marginal effect of grape-skin intervention was observed on plasma ascorbate levels. Intake of the experimental diet significantly reduced plasma vitamin C and plasma AAS in both groups. This effect was most pronounced in the particular week with no grape-skin extract addition. We speculate that grape-skin extract may have a sparing effect on vitamin C. The effects of the experimental diet may be partly ascribed to a low content of several fruit- and vegetable-related antioxidants like flavonoids and vitamin C and a relatively high content of carrot-derived antioxidants, such as carotenes.

The present study was carried out in order to investigate the in vivo biotransformation and excretion of the flavone, tangeretin, found in citrus fruits, by analysing urine and faeces samples from rats after repeated administration of 100 mg/kg body weight/day tangeretin. The formed metabolites were separated and identified by HPLC and the structures elucidated by LC/MS and 1H NMR. Ten new, major metabolites with intact flavonoid structure were identified. The metabolites identified were either demethylated or hydroxylated derivatives of the parent compound and metabolic changes were found primarily to occur in the 4' position of the B-ring. The total urinary excretion of tangeretin metabolites with intact flavan nucleus was about 11% of the administered daily dose.

A rapid and sensitive high-performance liquid chromatographic mass spectrometric (HPLC-MS) method is described for the determination and quantification of 12 dietary flavonoid glycosides and aglycons in human urine samples. Chromatographic separation of the analytes of interest was achieved by column-switching, using the first column (a Zorbax 300SB C-3 column) for sample cleanup and eluting the heart-cut flavonoid fraction onto the second column (a Zorbax SB C-18 column) for separation and detection by ultraviolet and atmospheric pressure chemical ionization MS using single ion monitoring in negative mode. The fragmentor voltage was optimized with regard to maximum abundance of the molecular ion and qualifier ions of the analytes. Calibration graphs were prepared for urine, and good linearity was achieved over a dynamic range of 2.5-1000 ng/mL. The inter- and intraassay coefficients of variation for the analysis of the 12 different flavonoids in quality control urine samples were 12.3% on average (range 11.0-13.7%, n = 24, reproducibility) and the repeatability of the assay were 5.0% (mean, range 0.1-14.8%, n = 12). A subset of 10 urine samples from a human dietary intervention study with high and low flavonoid content was analyzed, and the results are reported.

This intervention study was designed as cross-over (four women, one man) with three doses of black currant/apple (1:1) juice (750, 1000, and 1500 mL) for one week corresponding to an intake of 4.8, 6.4, and 9.6 mg quercetin per day. Urinary excretion of quercetin increased significantly with dose and with time. The fraction excreted in urine was constant 0.29-0.47%. Plasma quercetin did not change with juice intervention. Plasma ascorbate increased during intervention due to ascorbate from the juice. Total plasma malondialdehyde decreased with time during 1500 mL juice intervention. Plasma protein 2-adipic semialdehyde residues, increased with time and dose, and glutathione peroxidase increased with juice dose, whereas other selected markers of oxidative status did not change. These effects might be related to several components of the juice and cannot be attributed solely to its quercetin content.

Human exposure to genotoxic compounds present in ambient air has been studied using selected biomarkers in nonsmoking Danish bus drivers and postal workers. A large interindividual variation in biomarker levels was observed. Significantly higher levels of bulky carcinogen-DNA adducts (75.42 adducts/10(8) nucleotides) and of 2-amino-apidic semialdehyde (AAS) in plasma proteins (56.7 pmol/mg protein) were observed in bus drivers working in the central part of Copenhagen, Denmark. In contrast, significantly higher levels of AAS in hemoglobin (55.8 pmol/mg protein), malondialdehyde in plasma (0. 96 nmol/ml plasma), and polycyclic aromatic hydrocarbon (PAH)-albumin adduct (3.38 fmol/ microg albumin) were observed in the suburban group. The biomarker levels in postal workers were similar to the levels in suburban bus drivers. In the combined group of bus drivers and postal workers, negative correlations were observed between bulky carcinogen-DNA adduct and PAH-albumin levels (p = 0.005), and between DNA adduct and [gamma]-glutamyl semialdehyde (GGS) in hemoglobin (p = 0.11). Highly significant correlations were found between PAH-albumin adducts and AAS in plasma (p = 0.001) and GGS in hemoglobin (p = 0.001). Significant correlations were also observed between urinary 8-oxo-7, 8-dihydro-2'-deoxyguanosine and AAS in plasma (p = 0.001) and PAH-albumin adducts (p = 0.002). The influence of the glutatione S-transferase (GST) M1 deletion on the correlation between the biomarkers was studied in the combined group. A significant negative correlation was only observed between bulky carcinogen-DNA adducts and PAH-albumin adducts (p = 0.02) and between DNA adduct and urinary mutagenic activity (p = 0.02) in the GSTM1 null group, but not in the workers who were homozygotes or heterozygotes for GSTM1. Our results indicate that some of the selected biomarkers can be used to distinguish between high and low exposure to environmental genotoxins.

Radicals from the flavonoids quercetin, (+)-catechin, (+/-)-taxifolin and luteolin, and from all-rac-alpha-tocopherol have been generated electrochemically by one-electron oxidation in deaerated dimethylformamide (DMF), and characterised by electron spin resonance spectroscopy (ESR) after spin-trapping by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Simulations of the ESR spectrum based on estimated coupling constants of the spin-trapped quercetin radical, confirmed that this antioxidant radical is oxygen-centered. The complex mixture of radicals, quinoid intermediates and stable two-electron oxidation products, were for each antioxidant allowed to react with each of the four other antioxidants, and the progression of reaction followed by ESR after addition of DMPO, and the product solution further analysed by HPLC. All-rac-alpha-tocopherol was found to be most efficient in regenerating each of the other antioxidants from their oxidation products with a regeneration index (defined as moles regenerated of the oxidised phenolic antioxidant divided with moles of all-rac-alpha-tocopherol consumed) of 0.90+/-0.16 for quercetin, 0.48+/-0.11 for (+)-catechin, 0.48+/-0.06 for (+/-)-taxifolin and 0.50+/-0.10 for luteolin in equimolar 1.00 mM solution. Quercetin was found to have the highest regeneration index among the flavonoids: 0.88+/-0.13 for (+/-)-catechin, 0.41+/-0.03 for (+/-)-taxifolin and 0.41+/-0.02 for luteolin. The antioxidant hierarchy based on the reduction potentials determined by cyclic voltammetry under similar conditions (0.93 V for all-rac-alpha-tocopherol, 1.07 V for quercetin, 1.15 V for luteolin, 1.16V for (+)-catechin and 1.20 V for (+/-)-taxifolin) is compared with the observed over-all regeneration (34% for quercetin, 34% for (+)-catechin, 52% for (+/-)-taxifolin and 43% for luteolin by all-rac-alpha-tocopherol).

Background: Epidemiologic studies suggest that foods rich in flavonoids might reduce the risk of cardiovascular disease., Objective: Our objective was to investigate the effect of intake of flavonoid-containing black currant and apple juice on urinary excretion of quercetin and on markers of oxidative status., Design: This was a crossover study with 3 doses of juice (750, 1000, and 1500 mL) consumed for 1 wk by 4 women and 1 man corresponding to an intake of 4.8, 6.4, and 9.6 mg quercetin/d., Results: Urinary excretion of quercetin increased significantly with dose and with time. The fraction excreted in urine was 0.29-0.47%. Plasma quercetin did not change with juice intervention. Plasma ascorbate increased during intervention because of the ascorbate in the juice. Total plasma malondialdehyde decreased with time during the 1500-mL juice intervention, indicating reduced lipid oxidation in plasma. Plasma 2-amino-adipic semialdehyde residues increased with time and dose, indicating a prooxidant effect of the juice, whereas erythrocyte 2-aminoadipic semialdehyde and gamma-glutamyl semialdehyde concentrations, Trolox-equivalent antioxidant capacity, and ferric reducing ability of plasma did not change. Glutathione peroxidase activity increased significantly with juice dose., Conclusions: Urinary excretion of quercetin seemed to be a small but constant function of quercetin intake. Short-term, high intake of black currant and apple juices had a prooxidant effect on plasma proteins and increased glutathione peroxidase activity, whereas lipid oxidation in plasma seemed to decrease. These effects might be related to several components of the juice and cannot be attributed solely to its quercetin content.

Bulk electrolysis of the antioxidant flavonoids quercetin and kaempferol in acetonitrile both yield a single oxidation product in two-electron processes. The oxidation products are more polar than their parent compounds, with an increased molecular weight of 16g/mol, and were identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone and 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone for quercetin and kaempferol, respectively. Two-electron oxidation of the parent flavonoid is suggested to yield a 3,4-flavandione with unchanged substitution pattern in the A- and B-ring, which may rearrange to form the substituted 3(2H)-benzofuranone through the chalcan-trione ring-chain tautomer. The acidity of the 3-OH group is suggested to determine the fate of the flavonoid phenoxyl radical, originally formed by one-electron oxidation, as no well-defined oxidation product of luteolin (lacking the 3-OH group) could be isolated despite rather similar half-peak potentials: Ep/2 = 0.97V, 0.98 V and 1.17 V vs. NHE for quercetin, kaempferol and luteolin, respectively, as measured by cyclic voltammetry in acetonitrile.

A high-performance liquid chromatographic (HPLC) method is described for the determination of apigenin and the 4'-methylated derivative acacetin in human urine using column-switching and ultraviolet (UV) absorbance detection. Urine samples were enzymatically hydrolysed and solid-phase extracted prior to injection onto the HPLC system. Prior to elution of apigenin and the internal standard, 5,7,8-trihydroxyflavone, from the first column used for sample clean-up, the six-port valve was switched to the second column for analysis with UV detection. Detection of apigenin was precise and reproducible, with a limit of quantification of 10 ng ml(-1) urine. Detection and quantification of acacetin was linear down to 70 ng ml(-1) urine. The method has been successfully applied to determine the level of apigenin in 100 human urine samples from an intervention study with parsley.

A high-performance liquid chromatographic method is described for the determination of quercetin in human urine using column-switching and ultraviolet (UV) absorbance detection. Urine samples were enzymatically hydrolysed and solid-phase extracted prior to injection onto the high-performance liquid chromatography (HPLC) system. Prior to elution of quercetin and the internal standard, fisetin. from the first column used for sample clean-up, the six-port valve was switched to the second column for analysis with UV detection. Detection of quercetin was accurate and reproducible, with a detection limit of 5 ng/ml. The method was applied to determine the urinary level of quercetin in 120 samples from an intervention study with fruit juice.

Generation of reactive oxygen species in vivo results in oxidative-damage to cellular components, including proteins. Due to the relatively long half-lives of several blood proteins the cumulative formation of oxidatively damaged proteins might serve as a biomarker for reactive oxygen species formation. The most prominent sources of reactive oxygen species in vivo are site-specific metal ion-catalyzed reactions of the Fenton and Haber-Weiss types and the H2O2/peroxidase system. In vitro oxidation of L-tyrosine using a peroxidase or Cu++/H2O2 system gives rise to the formation of a highly fluorescent substance, bityrosine. High-performance liquid chromatography (HPLC) analysis of acid hydrolyzed serum albumin after oxidation with peroxidase/H2O2 or with Cu++/H2O2 showed that bityrosine had been formed whereas oxidation of this protein with Fe(III)/ascorbate did not result in the formation of bityrosine. Bityrosine could not be detected in human plasma proteins or haemoglobin with the detection limit of one pmol per mg protein.

Untreated and Aroclor 1254-pretreated male Wistar rats were given a single dose of 1.0 mg/kg body weight of randomly tritium-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (3H-PhIP) by oral intubation. Urine and faeces were collected at 24, 48 and 72 hours after dosing, and total radioactivity determined. At 2, 4, 6, 16, 26, 48 and 72 h, animals were killed and several organs, including liver, bladder, lungs, kidney, stomach, large and small intestines, heart, thigh muscles, spleen and blood were collected for DNA extraction and for determination of total radioactivity. Highest total radioactivity at 2 h was not unexpectedly observed in the stomach, small intestines and bladder, whereas radiolabels corresponding to approximately 2.5 nmol PhIP/g of kidney and liver showed the highest levels observed at 24 h. Several tissues, including blood, plasma, liver and muscles had a slightly bimodal time-distribution of radioactivity showing a second peak at 16-24 h. At 72 h after a single dose of PhIP, highest radioactivity was observed in the liver and the large intestine (0.4 nmol PhIP/g tissue), whereas most other organs, irrespective of pretreatment had levels at approximately 0.2 nmol/g of tissue. At earlier time points, Aroclor 1254-treated rats had lower amounts of radiolabel in all tissues. Radioactivity bound to DNA was determined by high sensitivity scintillation counting. In contrast to total radioactivity, DNA-associated radioactivity was generally higher in the Aroclor 1254-treated rats, most notably in the heart, but levels had decreased to approximately the same level in controls and in Aroclor 1254-treated rats at 72 h. DNA-binding was highest at 2-6 h after dosing, highest in the heart of Aroclor 1254-treated animals at 6 h (120 adducts/10(8) bases) followed by thigh muscle at 4-6 h (approximately 50 adducts/10(8) bases, irrespective of pretreatment). Levels were approximately 1.5-3 times lower in other organs at 2-6 h after dosing. At 72 h, radioactivity associated with DNA was again highest in the heart of Aroclor 1254-treated rats (20 adducts/10(8) bases) and 5-10 times lower in most other organs, approaching the detection limit. Total DNA was extracted from the livers of PhIP dosed rats at 4 ad 72 h. DNA was hydrolysed, affinity-concentrated, and analysed by liquid chromatography. A radiolabelled peak had identical retention time and UV-spectral characteristics as peaks isolated by affinity chromatography and HPLC of acid-hydrolysed synthetic PhIP-DNA and PhIP-deoxyguanosine adduct.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal mouse IgG1 and IgG3 antibodies were developed to the food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,4-f] quinoxaline (4,8-DiMeIQx) in order to make specific and sensitive detection and purification systems suitable for biological samples. The antibodies were developed with the strategy that cross-reaction with analogues modified in the N2-position was desirable. Competitive enzyme-linked immunosorbent assays (ELISA) with 50% inhibition by 0.4-6 pmol food mutagen were developed. The epitopes recognized by the antibodies have been characterized by ELISA using 52 synthetic analogues and metabolites of PhIP, 4,8-DiMeIQx, and other food mutagens. One of the anti-PhIP antibodies only recognizes PhIP and those PhIP-analogues which have minor modifications in the N2-amino group, whereas the other, 7B7-1, is less stringent and also recognizes several other modified metabolites, including bulky adducts at the N2-amino group e.g. the major guanine and deoxyguanosine adducts isolated from PhIP-modified DNA. The antibodies to DiMeIQx also recognize the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (4-MeIQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), and the corresponding quinolines (4-MeIQ and 8-MeIQ). Two of these antibodies only bind analogues with minor modifications in the free amino group, whereas analogues with major modifications in this position, including a deoxyguanosine adduct, react with the third antibody. Urine samples and faecal extracts from 3H-PhIP or 2-14C-DiMeIQx dosed rats were analysed by these ELISA assays, and high correlations between radioactivity and response in the ELISA assays were observed. Urine samples and faecal extracts from 3H-PhIP-dosed rats were purified on an affinity column containing the less stringent anti-PhIP antibody, 7B7-1. The affinity column was found by high performance liquid chromatography (HPLC) analysis to concentrate exclusively labelled material. This affinity column also bound PhIP-related materials from dilute samples of acid hydrolysed PhIP-DNA with high efficiency. Only approximately 40% of the 4,8-DiMeIQx related materials found in dilute acid hydrolysed samples of 4,8-DiMeIQx-DNA was bound by an affinity column containing the less stringent anti-4,8-DiMeIQx antibody, 2C5-1. We conclude that our anti-PhIP and anti-DiMeIQx antibodies can be used to determine the presence of these food mutagens and some of their activated or conjugated metabolites in complex biological samples.

The covalent binding of the mutagenic N2-hydroxy metabolite of the food mutagen 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx) to 2'-deoxynucleosides and DNA was investigated in vitro and in vivo. N2-Hydroxy-4,8-DiMeIQx reacted to a small extent spontaneously with 2-deoxyguanosine. However, acetylation of N2-hydroxy-4,8-DiMeIQx with acetic anhydride to form the N2-acetoxy derivative prior to reaction with 2-deoxyguanosine resulted in much higher yield of adduct. N2-Acetoxy-4,8-DiMeIQx did not form adducts with 2'-deoxyadenosine, 2'-deoxycytidine or 2'-deoxythymidine. The adduct formed between the N2-OH metabolite of 4,8-DiMeIQx and 2-deoxyguanosine was analysed by mass spectrometry and NMR spectroscopy and the structure of the adduct was shown to be N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx. N2-Acetoxy-4,8-DiMeIQx reacted with calf thymus DNA and formed a covalently bound 4,8-DiMeIQx residue, which could not be removed by repeated precipitations or solvent extractions. The 4,8-DiMeIQx-DNA was hydrolysed enzymatically with nuclease P1/acid phosphatase and HPLC analysis showed that 70% of the bound mutagen was recovered as N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx. An additional minor adduct accounting for approximately 15% of the bound mutagen showed UV spectral characteristics similar to N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx and is probably an undigested oligomer. 32P-Postlabelling analysis of calf thymus DNA modified with 4,8-DiMeIQx in vitro and liver DNA from rats dosed with 50 mg/kg 4,8-DiMeIQx showed a similar adduct pattern. In both samples N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx accounted for 60-70% of the bound mutagen. Thus, these results show that 4,8-DiMeIQx similar to other heterocyclic amines form adducts with C-8 of guanine both in vitro and in vivo via its N2-OH metabolite.

Cancer-protective factors are present in several fruits, vegetables and commonly used spices and herbs. They can be divided into several different groups, based on their chemical structure, e.g. polyphenols, thiols, carotenoids and retinoids, carbohydrates, trace metals, terpenes, tocopherols and degradation products of glucosinolates (i.e. isothiocyanates, indoles and dithiothiols) and others. Among each of these groups of compounds are substances, which may exert their cancer-protective action by more than one biochemical mechanism. The biochemical processes of carcinogenesis are still not known in detail and probably varies with the cancer disease in question. Accordingly, the description of the biochemical backgrounds for the actions of cancer-protective factors must be based on a simplified model of the process of carcinogenesis. The model used in this presentation is a generalised initiation-promotion-conversion model, in which initiators are thought to be directly or indirectly genotoxic, promoters are visualised as substances capable of inferring a growth advantage on initiated cells and converters are believed to be genotoxic, e.g. mutagens, clastogens, recombinogens or the like. Experimental evidence for the mechanisms of action of cancer-protective agents in fruits and vegetables that protect against initiation include the scavenging effects of polyphenols on activated mutagens and carcinogens, the quenching of singlet oxygen and radicals by carotenoids, the antioxidant effects of many compounds including ascorbic acid and polyphenols, the inhibition of activating enzymes by some flavonols and tannins, the induction of oxidation- and of conjugation (protective) enzymes by indoles, isothiocyanates and dithiothiones, the shielding of sensitive structures by some polyphenols and the stimulation of DNA-repair exerted by sulphur-containing compounds. Mechanisms at the biochemical level in anti-promotion include the antioxidant effects of carotenoids and the membrane stabilizing effects reported with polyphenols, the inhibition of proteases caused by compounds from soybeans, the stimulation of immune responses seen with carotenoids and ascorbic acid and the inhibition of ornithine decarboxylase by polyphenols and carotenoids. A few inhibitors of conversion have been identified experimentally, and it can be argued on a theoretical basis, that many inhibitors of initiation should also be efficient against conversion. The mechanisms of anticarcinogenic substances in fruits and vegetables are discussed in the light of cancer prevention and inhibition.