The safety of a Kluyveromyces lactis strain lineage for enzyme production.

Kluyveromyces lactis is broadly considered as a safe yeast in food and a suitable organism for the production of food enzymes. The K. lactis enzyme production strains of DSM are used to produce a variety of enzymes, for example beta-galactosidase (lactase), chymosin and esterase. All of these production strains are derived from the same lineage, meaning they all originate from the same ancestor strain after classical mutagenesis and/or genetic engineering. Four different enzyme preparations produced with strains within this lineage were toxicologically tested. These enzyme preparations were nontoxic in repeated-dose oral toxicity studies performed in rats and were non-genotoxic in vitro. These studies confirm the safety of the DSM K. lactis strains as a production platform for food enzymes, as well as the safety of the genetic modifications made to these strains through genetic engineering or classical mutagenesis. The outcome of the toxicity studies can be extended to other enzyme preparations produced by any strain from this lineage through read across. Therefore, no new toxicity studies are required for the safety evaluation, as long as the modifications made do not raise safety concerns. Consequently, this approach is in line with the public ambition to reduce animal toxicity studies.

The exposome concept in a human nutrigenomics study: evaluating the impact of exposure to a complex mixture of phytochemicals using transcriptomics signatures.

The application of transcriptome analyses in molecular epidemiology studies has become a promising tool in order to evaluate the impact of environmental exposures. These analyses have a great value in establishing the exposome, the totality of human exposures, both by identifying the chemical nature of the exposures and the induced molecular responses. Transcriptomic signatures can be regarded as biomarker of exposure as well as markers of effect which reflect the interaction between individual genetic background and exposure levels. However, the biological interpretation of modulated gene expression profiles is a challenging task and translating affected molecular pathways into risk assessment, for instance in terms of cancer promoting or disease preventing responses, is a far from standardised process. Here, we describe the in-depth analyses of the gene expression responses in a human dietary intervention in which the interaction between genotype and exposure to a blueberry-apple juice containing a complex mixture of phytochemicals is investigated. We also describe how data on differences in genetic background combined with different effect markers can provide a better understanding of gene-environment interactions. Pathway analyses of differentially expressed genes in combination with gene were used to identify complex but strong changes in several biological processes like immune response, cell adhesion, lipid metabolism and apoptosis. These observed changes may lead to upgraded growth control, induced immunity, reduced platelet aggregation and activation, diminished production of reactive oxidative species by platelets, blood glucose homeostasis, regulation of blood lipid levels and increased apoptosis. Our findings demonstrate that applying transcriptomics to well-controlled human dietary intervention studies can provide insight into mechanistic pathways involved in disease prevention by dietary factors.

Can transcriptomics provide insight into the chemopreventive mechanisms of complex mixtures of phytochemicals in humans?

Blueberries contain relatively large amounts of different phytochemicals, which are suggested to have chemopreventive properties, but little information is available on the underlying molecular modes of action. This study investigates whole genome gene expression changes in lymphocytes of 143 humans after a 4-week blueberry-apple juice dietary intervention. Differentially expressed genes and genes correlating with the extent of antioxidant protection were identified in four subgroups. The magnitude of the preventive effect after the intervention differed between these four subgroups. Furthermore, subjects in two groups carried genetic polymorphisms that were previously found to influence the chemopreventive response. Pathway analysis of the identified genes showed strong but complex gene expression changes in pathways signaling for apoptosis, immune response, cell adhesion, and lipid metabolism. These pathways indicate increased apoptosis, upgraded growth control, induced immunity, reduced platelet aggregation and activation, blood glucose homeostasis, and regulation of fatty acid metabolism. Based on these observations, we hypothesize that combining transcriptomic data with phenotypic markers of oxidative stress may provide insight into the relevant cellular processes and genetic pathways, which contribute to the antioxidant response of complex mixtures of phytochemicals, such as found in blueberry-apple juice.

Genetic polymorphisms in catalase and CYP1B1 determine DNA adduct formation by benzo(a)pyrene ex vivo.

Genetic polymorphisms can partially explain the large inter-individual variation in DNA adduct levels following exposure to polycyclic aromatic hydrocarbons. Effects of genetic polymorphisms on DNA adduct formation are difficult to assess in human studies because exposure misclassification attenuates underlying relationships. Conversely, ex vivo studies offer the advantage of controlled exposure settings, allowing the possibility to better elucidate genotype-phenotype relationships and gene-gene interactions. Therefore, we exposed lymphocytes of 168 non-smoking volunteers ex vivo to the environmental pollutant benzo(a)pyrene (BaP) and BaP-related DNA adducts were quantified. Thirty-four genetic polymorphisms were assessed in genes involved in carcinogen metabolism, oxidative stress and DNA repair. Polymorphisms in catalase (CAT, rs1001179) and cytochrome P450 1B1 (CYP1B1, rs1800440) were significantly associated with DNA adduct levels, especially when combined. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) analysis in a subset of 30 subjects revealed that expression of catalase correlated strongly with expression of CYP1B1 (R = 0.92, P < 0.001). To further investigate the mechanism by which catalase influences CYP1B1 and how they simultaneously affect BaP-related DNA adduct levels, catalase expression was transiently knocked down in the human lung epithelial cell line A549. Although catalase knockdown did not immediately change CYP1B1 gene expression, recovery of catalase expression 8 h after the knockdown coincided with a 2.2-fold increased expression of CYP1B1 (P < 0.05). We conclude that the genetic polymorphism in the promoter region of CAT may determine the amount and activity of catalase, which may subsequently regulate the expression of CYP1B1. As a result, both genetic polymorphisms modulate DNA adduct levels in lymphocytes by BaP ex vivo.

Modulation of nucleotide excision repair in human lymphocytes by genetic and dietary factors.

Gene-environment interactions determine inter-individual variations in nucleotide excision repair (NER) capacity. Oxidative stress was previously found to inhibit NER, thus supplementation with dietary antioxidants could prevent this inhibition, especially in genetically susceptible subjects. To study the effects of genetic polymorphisms in NER-related genes and dietary intake of antioxidants on an individual's NER capacity, lymphocytes of 168 subjects were isolated before and after a 4-week blueberry and apple juice intervention. Twelve genetic polymorphisms in NER genes XPA, XPC, ERCC1, ERCC2, ERCC5, ERCC6 and RAD23B were assessed by multiplex PCR with single base extension. Based on specific genotype combinations, a subset of individuals (n 36) was selected for phenotypical assessment of NER capacity, which was significantly affected by the total sum of low-activity alleles (P = 0.027). The single polymorphism XPA G23A was the strongest predictor of NER capacity (P = 0.002); carriers of low-activity alleles AA had about three times lower NER capacity than XPA GG carriers. NER capacity assessed before and after intervention correlated significantly (R(2) 0.69; P < 0.001), indicating that inter-individual differences in NER capacity are maintained over 4 weeks. Although the intervention increased plasma trolox equivalent antioxidant capacity from 791 (SE 6.61) to 805 (SE 7.90) microm (P = 0.032), on average it did not affect NER capacity. Nonetheless, carriers of twelve or more low-activity alleles seemed to benefit from the intervention (P = 0.013). Among these, carriers of the variant allele for RAD23B Ala249Val showed improved NER capacity upon intervention (P = 0.020). In conclusion, improved NER capacity upon dietary intervention was detected in individuals carrying multiple low-activity alleles. The XPA G23A polymorphism might be a predictor for NER capacity.

Antioxidative and antigenotoxic properties of vegetables and dietary phytochemicals: the value of genomics biomarkers in molecular epidemiology.

There is considerable evidence that consumption of fruits and vegetables may contribute to the prevention of cancer. It is however remarkable that evidence for such a preventive action arising from mechanistic studies is becoming stronger, whereas results of some recent prospective studies are less convincing. This apparent discrepancy may be overcome, or at least understood, by introducing molecular markers in future epidemiological studies, taking modulation of molecular processes as well as genetic variability in human populations into account. Both human and animal studies demonstrated that vegetable intake modulates gene expression in the gastrointestinal tract of many genes involved in biological pathways in favor of cancer risk prevention. Gene sets identified in this type of studies can be further evaluated, linked to the biological effects of phytochemicals and developed into biomarkers for larger human studies. Human dietary intervention studies have demonstrated that, apart from target tissues, also peripheral lymphocytes can be used for biomonitoring of chemopreventive effects. Transcriptomic responses and metabolite profiling may link phenotypic markers of preventive effects to specific molecular processes. The use of genomics techniques appears to be a promising approach to establish mechanistic pathways involved in chemoprevention by phytochemicals, particularly when genetic variability is taken into account.

In vitro and ex vivo anti-inflammatory activity of quercetin in healthy volunteers.

OBJECTIVE: Quercetin, a commonly occurring flavonoid and well known antioxidant, has been suggested to possess other beneficial activities. The present study investigated the possible anti-inflammatory effects of physiologically attainable quercetin concentrations. METHODS: The effects of quercetin were tested in vitro, i.e., added to blood in the test tube, and ex vivo and in vivo, i.e., in blood taken after 4 wk of administration of quercetin in an intervention study. RESULTS: Quercetin dose-dependently inhibited in vitro lipopolysaccharide-induced tumor necrosis factor-alpha production in the blood of healthy volunteers. At a concentration of 1 muM, quercetin caused a 23% reduction. The in vitro lipopolysaccharide-induced interleukin-10 production remained unaffected by quercetin. A 4-wk quercetin intervention resulted in a significant increase in plasma quercetin concentration. The supplementation also increased total plasma antioxidant status but did not affect glutathione, vitamin C, and uric acid plasma concentrations. Basal and ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha levels were not altered by the intervention. CONCLUSION: The present study shows that quercetin increases antioxidant capacity in vivo and displays anti-inflammatory effects in vitro, but not in vivo or ex vivo, in the blood of healthy volunteers. This lack of effect is probably due to their low cytokine and high antioxidant levels at baseline, indicating that neither inflammation nor oxidative stress is present. Only in people with increased levels of inflammation and oxidative stress, e.g., patients with a disease of which the pathology is associated with these two processes, might antioxidant supplementation be fruitful.

Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro.

Antioxidants play a vital role in the cellular protection against oxidative damage. Quercetin is a well-investigated antioxidant and known to be able to protect against cellular oxidative DNA damage. In this study, we tried to relate the protection by quercetin pre-treatment against oxidative DNA damage in human leucocytes in vitro to the interaction of quercetin in solution with hydroxyl and superoxide anion radicals as measured by electron spin resonance (ESR) spectrometry, using DMPO as a spin trap. Further, scavenging capacity of quercetin-treated leucocytes in vitro was evaluated by ESR spectrometry. Quercetin appears capable of protecting human leucocytes against oxidative DNA damage caused by hydrogen peroxide in a dose-dependent manner. The protection of leucocytes against superoxides is ambiguous. Incubation concentrations of quercetin (1, 10, and 50 microM) reduced levels of superoxide-induced oxidative DNA damage, while at 100 microM the amount of damage was increased. These results are supported by ESR-findings on quercetin in solution, also showing a prooxidant effect at 100 microM. ESR spectroscopy showed rate constant values for the reaction kinetics of quercetin in lowering iron-dependent hydroxyl radical formation and NADH-dependent superoxide anion formation of respectively 3.2 x 10(12)M(-1)s(-1) and 1.1 x 10(4)M(-1)s(-1). This shows that quercetin is a more potent inhibitor of hydroxyl radical formation than a scavenger of superoxide anions.

GSTM1 and GSTT1 polymorphism influences protection against induced oxidative DNA damage by quercetin and ascorbic acid in human lymphocytes in vitro.

UNLABELLED: Antioxidants are of major importance in the protection against cellular oxidative damage caused by endogenous as well as exogenous free radicals. This study aims to establish the impact of genetic polymorphisms in GSTM1 and GSTT1, which encode for enzymatic antioxidative defence, on H(2)O(2)-induced oxidative DNA damage and on the effectiveness of quercetin and ascorbic acid in preventing this induced damage in human lymphocytes. Lymphocytes from 12 healthy volunteers were pre-incubated either with 10 microM of quercetin or with 10 microM of ascorbic acid, and exposed to 25 microM H(2)O(2) for 1h. The induction of oxidative DNA damage was quantified using the Comet assay. Genotyping of these 12 subjects showed that six individuals were GSTM1+ and six were GSTM1-; eight were GSTT1+ and four GSTT1-. RESULTS: Baseline levels of oxidative DNA damage did not differ between GSTM1 or GSTT1 variants and their respective wild types. Also with respect to ex vivo induced levels of oxidative DNA damage, no significant difference was seen between variants and wild types of both genotypes. The protection against H(2)O(2)-induced oxidative DNA damage by quercetin was significantly higher in GSTT1 wild types than in GSTT1 variants (57% and 9% decrease, respectively; p=0.01); furthermore, GSTT1 wild types were protected against induced oxidative DNA damage by ascorbic acid pre-incubation while GSTT1 variants showed an increase of damage (16% decrease vs. 91% increase; p=0.01). For GSTM1 variants and wild types, observed differences in protective effects of quercetin or ascorbic acid were not statistically significant. Overall, quercetin proves to be better in protecting human lymphocytes in vitro against oxidative DNA damage upon H(2)O(2) challenge than ascorbic acid.

Impact of multiple genetic polymorphisms on effects of a 4-week blueberry juice intervention on ex vivo induced lymphocytic DNA damage in human volunteers.

Consumption of fruits and vegetables has been associated with a decrease in cancer incidence and cardiovascular disease, presumably caused by antioxidants. We designed a human intervention study to assess antioxidative and possible anti-genotoxic properties of fruit-borne antioxidants. We hypothesized that individuals bearing genetic polymorphisms for genes related to quercetin metabolism, benzo[a]pyrene metabolism, oxidative stress and DNA repair differ in their response to DNA protective effects of increased antioxidant intake. In the present study, 168 healthy volunteers consumed a blueberry/apple juice that provided 97 mg quercetin and 16 mg ascorbic acid a day. After a 4-week intervention period, plasma concentrations of quercetin and ascorbic acid and trolox equivalent antioxidant capacity (TEAC) were significantly increased. Further, we found 20% protection (P < 0.01) against ex vivo H(2)O(2)-provoked oxidative DNA damage, measured by comet assay. However, the level of ex vivo induced benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts was 28% increased upon intervention (P < 0.01). Statistical analysis of 34 biologically relevant genetic polymorphisms revealed that six significantly influenced the outcome of the intervention. Lymphocytes from individuals bearing variant genotype for Cyp1B1 5 seemed to benefit more than wild-types from DNA damage-protecting effects upon intervention. Variants for COMT tended to benefit less or even experienced detrimental effects from intervention. With respect to GSTT1, the effect is ambiguous; variants respond better in terms of intervention-related increase in TEAC, but wild-types benefit more from its protecting effects against oxidative DNA damage. We conclude that genotyping for relevant polymorphisms enables selecting subgroups among the general population that benefit more of DNA damage-modulating effects of micronutrients.

Protection by quercetin and quercetin-rich fruit juice against induction of oxidative DNA damage and formation of BPDE-DNA adducts in human lymphocytes.

Flavonoids are claimed to protect against cardiovascular disease, certain forms of cancer and ageing, possibly by preventing initial DNA damage. Therefore, we investigated the protective effects of the flavonoid quercetin against the formation of oxidative DNA damage and bulky DNA adducts in human lymphocytes, both in vitro and ex vivo. First, human lymphocytes were pre-incubated with various concentrations of quercetin, followed by incubation with hydrogen peroxide; protection against oxidative DNA damage was evaluated by use of the single-cell gel electrophoresis (Comet) assay. Second, quercetin-treated human lymphocytes were challenged by treatment with benzo(a)pyrene (B(a)P), and BPDE-DNA adduct formation was measured by (32)P-postlabelling. Third, in a pilot study, lymphocytes from female volunteers who consumed a quercetin-rich blueberry/apple juice mixture for four weeks, were treated ex vivo with an effective dose of H(2)O(2) and benzo(a)pyrene, respectively, at three different time points, i.e. before (t=0 weeks), during (t=2 weeks) and after (t=4 weeks) the intervention. Results in vitro: a significant dose-dependent protection by quercetin against both the formation of oxidative DNA damage (p<0.01) and of BPDE-DNA adducts (p<0.05) was observed. Results in vivo: four weeks of juice intervention led to a significant increase in the total antioxidant capacity of plasma, as reflected by the increase of the TEAC value from 773 microM trolox equivalent at t=0 to 855 microM at t=4 weeks (p=0.04) and an increase in plasma quercetin content from 5.0 to 10.6 nM (p=0.03). After intervention, the level of oxidative damage upon ex vivo exposure to H(2)O(2) was non-significantly (p=0.07) decreased by 41%, and the BPDE-DNA adduct level induced ex vivo was non-significantly decreased by 11%. The combination of our findings in vitro and ex vivo provides evidence that quercetin is able to protect against chemically induced DNA damage in human lymphocytes, which may underlie its suggested anticarcinogenic properties.

Measurement of cyclobutane thymidine dimers in melanocytic nevi and surrounding epidermis in human skin in situ.

The numbers of melanocytic nevi, localized benign proliferations of melanocytes, have been shown to be associated with an increased risk for development of melanoma. In the present study we have developed an alternative post-labelling method for determination of levels of cyclobutane thymidine dimers (T=T) as dinucleotides at sensitivities sufficient for analysis of human skin samples. Using the developed method, the induction of T=T was determined in melanocytic nevi in situ and surrounding skin, obtained from seven subjects, after exposure to solar simulating radiation. The T=T level in nevi was found to be 1- to 4.5-fold lower than that in surrounding skin and the difference was statistically significant (Student's t-test, P < 0.05).

Interactions between flavonoids and proteins: effect on the total antioxidant capacity.

Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect of the interaction between tea flavonoids and proteins on the antioxidant capacity was examined. Their separate and combined antioxidant capacities were measured with the Trolox equivalent antioxidant capacity (TEAC) assay. It was observed that the antioxidant capacity of several components of green and black tea with alpha-, beta-, and kappa-casein or albumin is not additive; that is, a part of the total antioxidant capacity is masked by the interaction. This masking depends on both the protein and the flavonoid used. Components in green and black tea, which show the highest masking in combination with beta-casein, are epigallocatechin gallate and gallic acid. The results demonstrate that the matrix influences the efficacy of an antioxidant.