The natural compound ascorbigen modulates NADPH-quinone oxidoreductase (NQO1) mRNA and enzyme activity levels in cultured liver cells and in laboratory rats.

Ascorbigen (ABG) is a natural compound that represents a breakdown product of the glucosinolates that are present in Brassica vegetables. It is postulated that ABG may have anticarcinogenic activity; however, the underlying molecular and cellular mechanisms are largely unknown. In the present study we investigated the effect of ABG on the mRNA and enzyme activity levels of NADPH-quinone oxidoreductase (NQO1), which is centrally involved in the detoxification of xenobiotics, in cultured liver cells and in rats. The mRNA levels of NQO1 showed an increase of up to 100% in cultured liver cells (HepG2) following incubation with different concentrations of ABG (3-100 micromol/l) compared to control cells. Furthermore, NQO1 activity was elevated (up to 20%) by ABG treatment. The in vitro results were confirmed in rats who received either 5 mg/day ABG or vehicle for 7 days. Significantly higher mRNA (a 90% increase) and enzyme activity levels (a 40% increase) of NQO1 were detected in the liver of ABG-treated rats as compared to control animals. Current data indicate that ABG is a moderate inducer of the phase II enzyme NQO1, both in cultured hepatocytes and in vivo.

Beta-carotene interaction with NNK in the AJ-mouse model: effects on cell proliferation, tumor formation and retinoic acid responsive genes.

We studied the influence of beta-carotene on the tobacco smoke carcinogen 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor development in the A/J-mouse model. The normally low beta-carotene absorption was facilitated with a diet enriched in fat and bile salt, resulting in plasma and lung tissue levels similar to humans. beta-Carotene enhanced NNK-induced early bronchial cell proliferation, however, this effect was not predictive for later tumor development. Tumor multiplicity was not significantly affected by beta-carotene, neither in carcinogen-initiated nor in uninitiated mice, and regardless of dose and time point of supplementation during tumor development. RARbeta isoform and CYP26 gene expression levels analyzed by quantitative RT-PCR were weakly, but significantly, inversely correlated and showed evidence for altered retinoid signaling and catabolism in the lungs of NNK-initiated, beta-carotene supplemented mice. However, this interaction did not translate into enhanced tumor multiplicity. These results indicate that impaired retinoid signaling is not likely a key factor in lung tumorigenesis in this mouse model.

The effect of beta-carotene and its derivatives on cytotoxicity, differentiation, proliferative potential and apoptosis on the three human acute leukemia cell lines: U-937, HL-60 and TF-1.

The influence of beta-carotene (BC) and its derivatives on differentiation, proliferation and apoptosis in three human acute leukemia cell lines was studied. We investigated: (i) the cellular uptake of BC, (ii) the cytotoxicity, (iii) the effect on cell cycle progression and/or apoptosis. The dose- and time-dependent pattern of cellular BC uptake in all studied cell lines was seen. We did not observe any cytotoxic effect of BC and ATRA in the chosen concentrations. There was only limited effect of BC on gene expression. The microarrray analysis of U-937 cell line exposed to BC for 72 h showed an increased expression of BAX gene. This finding was confirmed by real-time Q-PCR analysis, and supported by a flow cytometry apoptosis tests. We did not observe any influence of studied components on cellular proliferation. The induction of differentiation after incubation with ATRA in HL-60 cells was noted. The induction of cellular apoptosis by BC was seen in all studied cell lines. We demonstrated that BC used in the concentrations achievable in vivo does not affect the proliferation and differentiation process of the studied leukemic cell lines, but can influence and enhance the apoptosis by modulating the expression of the regulatory genes.

beta-Carotene interferes with ultraviolet light A-induced gene expression by multiple pathways.

Ultraviolet light A (UVA) exposure is thought to cause skin aging mainly by singlet oxygen ((1)O(2))-dependent pathways. Using microarrays, we assessed whether pre-treatment with the (1)O(2) quencher beta-carotene (betaC; 1.5 microM) prevents UVA-induced gene regulation in HaCaT human keratinocytes. Downregulation of growth factor signaling, moderate induction of proinflammatory genes, upregulation of immediate early genes including apoptotic regulators and suppression of cell cycle genes were hallmarks of the UVA effect. Of the 568 UVA-regulated genes, betaC reduced the UVA effect for 143, enhanced it for 180, and did not interact with UVA for 245 genes. The different interaction modes imply that betaC/UVA interaction involved multiple mechanisms. In unirradiated keratinocytes, gene regulations suggest that betaC reduced stress signals and extracellular matrix (ECM) degradation, and promoted keratinocyte differentiation. In irradiated cells, expression profiles indicate that betaC inhibited UVA-induced ECM degradation, and enhanced UVA induction of tanning-associated protease-activated receptor 2. Combination of betaC-promoted keratinocyte differentiation with the cellular "UV response" caused synergistic induction of cell cycle arrest and apoptosis. In conclusion, betaC at physiological concentrations interacted with UVA effects in keratinocytes by mechanisms that included, but were not restricted to (1)O(2) quenching. The retinoid effect of betaC was minor, indicating that the betaC effects reported here were predominantly mediated through vitamin A-independent pathways.

Beta-carotene inhibits UVA-induced matrix metalloprotease 1 and 10 expression in keratinocytes by a singlet oxygen-dependent mechanism.

UVA exposure causes skin photoaging by singlet oxygen (1)O(2)-mediated induction of, e.g., matrix metalloproteases (MMPs). We assessed whether pretreatment with beta-carotene, a (1)O(2) quencher and retinoic acid (RA) precursor, interferes with UVA-induced gene regulation. HaCaT keratinocytes were precultured with beta-carotene at physiological concentrations (0.5, 1.5, and 3.0 microM) prior to exposure to UVA from a Hönle solar simulator (270 kJ/m(2)). HaCaT cells accumulated beta-carotene in a time- and dose-dependent manner. UVA irradiation massively reduced the cellular beta-carotene content. Beta-carotene suppressed UVA-induction of MMP-1, MMP-3, and MMP-10, three major matrix metalloproteases involved in photoaging. We show that regulation by not only MMP-1, but also MMP-10, involves (1)O(2)-dependent mechanisms. Beta-carotene dose-dependently quenched (1)O(2)-mediated induction of MMP-1 and MMP-10. Thus, as in chemical solvent systems, beta-carotene quenches (1)O(2) also in living cells. Vitamin E did not cooperate with beta-carotene to further inhibit MMP induction. HaCaT cells produced weak retinoid activity from beta-carotene, as demonstrated by mild upregulation of RAR beta and activation of an RARE-dependent reporter gene. Beta-carotene did not regulate the genes encoding other RARs, RXRs, or the two beta-carotene cleavage enzymes. These results demonstrate that beta-carotene acts photoprotectively, and that this effect is mediated by (1)O(2) quenching.

Rapid determination of 25-hydroxy vitamin D3 in swine tissue using an isotope dilution HPLC-MS assay.

A rapid method for quantification of 25-hydroxy vitamin D3 in different swine tissues based on isotope dilution HPLC-MS has been developed and validated. Six times deuterated analyte is used as internal standard. The method is fast and can be performed with only 1g sample. Sample preparation for kidney, liver, muscle and spleen requires only homogenisation and extraction with methanol. An additional enzymatic digest is required for skin, and clean-up of the extract by solid-phase extraction (SPE) is used for adipose tissue and skin. The lower limit of detection varies from 1 ng/g (muscle) to 5 ng/g (adipose and skin). The method has been successfully applied to various tissue samples of pigs fed for 119 days either 2000 IU of vitamin D3 or 50 microg of 25-hydroxy vitamin D3 per kg feed. For animals ingesting 25-OH-D3 supplements the highest tissue contents were observed in the skin (24.8+/-3.5 ng/g), followed by kidney (14.2+/-1.5 ng/g), liver and muscle (5.7+/-0.6 ng/g). The 25-OH-D3 content in the skin was significantly higher in animals ingesting 2000 IU/kg of vitamin D3 (39.5+/-13.4 ng/g). Levels in selected tissues of some animals were below the lower limit of quantification. No measurable amounts of 25-OH-D3 were found in spleen, abdominal fat and subcutaneous fat of the animals of both groups as well as in the liver, kidney and muscle of the animals ingesting 2000 IU/kg of vitamin D3.

Transcriptomics does not show adverse effects of beta-carotene in A/J mice exposed to smoke for 2 weeks.

Beta-carotene (betaC) supplementation in smokers was unexpectedly associated with increased incidence of lung cancer versus smoking alone. We performed a study in A/J mice to explore possible betaC/cigarette smoke (CS) interactions potentially influencing lung cancer risk in smokers. A/J mice received a diet containing 120 or 600 ppm betaC for six weeks, and exposed to mainstream CS (140 mg total suspended particulates/m(3)) during the last two weeks. Lung transcriptomics analysis revealed that CS induced drug metabolism, oxidative stress, extracellular matrix (ECM) degradation, inflammation markers, and apoptosis. betaC reduced CS-induced inflammation markers and ECM degradation. betaC modulated the CS effect on apoptosis without a clear pro- or anti-apoptotic trend. betaC alone induced only minor changes of gene expression. In conclusion, betaC/CS interactions caused gene regulations in lungs. CS was the main effector. The gene regulations overall did not indicate that betaC exacerbated CS effects. Dose-dependency of betaC effects was minor and not detectable by genome-wide data mining.

beta-carotene-induced changes in RARbeta isoform mRNA expression patterns do not influence lung adenoma multiplicity in the NNK-initiated A/J mouse model.

A number of epidemiological studies have reported associations of beta-carotene plasma levels or intake with decreased lung cancer risk. However, intervention studies in smokers reported increased lung tumor rates after high long-term beta-carotene supplementation. For insight into these conflicting results, we studied the influence of beta-carotene on tobacco smoke carcinogen-induced lung cancer development in the A/J-mouse using 4-(N-Methyl-N-nitro samino)-1-(3-pyridyl)-1-butanone (NNK) as the initiator and lung adenoma multiplicity as the functional endpoint. Gene regulation of the putative tumor suppressor RARbeta in mouse lung was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for its relevance in predicting the endpoint of lung cancer. A/J-mice achieved plasma beta-carotene levels of up to 3 micromol/L within 4 wk and up to 6 micromol/L after 6 mo of supplementation on a diet modified to enhance beta-carotene absorption. Despite high lung beta-carotene concentrations of up to 6 micromol/kg, tumor multiplicity was not significantly affected by the beta-carotene treatment, either in carcinogen-initiated or non-initiated mice, and was unrelated to beta-carotene dose and the time point of treatment during cancer formation. Tumor multiplicity did not correlate with beta-carotene plasma levels in NNK-treated animals. All RARbeta isoforms were significantly suppressed in the lungs of NNK- and NNK plus high dose beta-carotene-treated animals. However, the number of tumors per mouse did not correlate with the RARbeta-isoform expression levels. beta-carotene alone after 3 mo of supplementation mildly but significantly increased levels of RARbeta1, beta2, and beta4. This increase persisted for 6 mo for RARbeta2 and beta4. In summary, we found no effect of beta-carotene on tumor formation in the NNK-initiated A/J-mouse lung cancer model with respect to dose or time point of treatment. beta-Carotene-induced changes in RARbeta isoform gene expression levels were not predictive for the number of lung tumors but were indicative of intact beta-carotene metabolism and persistent sensitivity to retinoic acid in the mice. Down-regulation of RARbeta in NNK-induced adenoma-bearing lungs was similar to that observed in human lung cancer and further confirms the A/J-mouse as a valuable model for lung carcinogenesis.

Beta-carotene and apocarotenals promote retinoid signaling in BEAS-2B human bronchioepithelial cells.

High dose beta-carotene supplementation of smokers was associated with increased lung cancer risk in two intervention trials. It was proposed that generation of apocarotenals in smoke-exposed lungs impaired retinoic acid (RA) signaling, leading to squamous metaplasia and cell proliferation. To test this, we compared RA target gene regulation by retinoids, apocarotenals or beta-carotene by transcriptomics in BEAS-2B cells cultured to promote squamous differentiation. Retinoids, beta-carotene as well as apocarotenals induced known RA target genes. Retinoids upregulated involucrin, indicating that retinoids did not rescue BEAS-2B cells from squamous differentiation. Muc5AC, a marker for mucous differentiation, was transiently induced. beta-Carotene and apocarotenals less strongly induced involucrin and did not induce muc5AC. In summary, apocarotenals or beta-carotene upregulated RA target genes suggesting promotion, not inhibition, of RA signaling in BEAS-2B cells. Furthermore, apocarotenals and beta-carotene regulated gene expression independently of RA signaling. Squamous differentiation is not unequivocally linked to RA deficiency in BEAS-2B cells.

Betacarotene supplementation protects from photoaging-associated mitochondrial DNA mutation.

Mutations of mitochondrial DNA accumulate during normal aging and can be detected at elevated levels in skin prematurely aged by chronic exposure to ultraviolet (UV) light (photoaging). In normal human fibroblasts, we have previously demonstrated that mtDNA deletions are induced by repetitive exposure to sublethal doses of UVA radiation mediated through singlet oxygen. Betacarotene is a known quencher of ROS and singlet oxygen in particular, and it is widely applied in photoprotective compounds. Therefore we investigated whether in our in vitro system, betacarotene is capable of protecting from the induction of photoaging-associated mtDNA deletions. All-E (trans) betacarotene was tested at doses from 0.25 to 3.0 microM for uptake into cells as well as its protective capacity. Assessment of cellular uptake of all-E betacarotene measured by HPLC revealed a dose dependent increase of intracellular concentrations, as well as an increase in oxidative metabolites. UVA-exposure led to a decrease of all-E-betacarotene, its Z-isomers and oxidative metabolites. Assessment of mtDNA deletions by PCR revealed reduced levels of mtDNA mutagenesis in cells coincubated with betacarotene at concentrations of 0.5 microM and higher. Taken together, these results indicate that betacarotene (i) is taken up into the cell in a dose dependent manner, (ii) interacts with UVA radiation in the cell and (iii) shows protective properties from the induction of a photoaging-associated mtDNA mutation.

Feedback regulation of beta,beta-carotene 15,15'-monooxygenase by retinoic acid in rats and chickens.

beta,beta-Carotene 15,15'-monooxygenase (formerly termed beta,beta-carotene 15,15'-dioxygenase, EC 1.13.11.21) catalyzes the conversion of provitamin A carotenoids to retinal in vertebrate tissues. In the present study, we investigated whether preformed vitamin A or beta-carotene and its direct metabolites can regulate the enzyme activity in vivo. We found dose-dependent decreases in intestinal beta,beta-carotene monooxygenase activity after oral administration to rats of retinyl acetate (up to -79%), beta-carotene (up to -79%), apo-8'-carotenal (up to -56%), all-trans retinoic acid (up to -88%), and 9-cis retinoic acid (up to -67%). Liver beta,beta-carotene 15,15'-monooxygenase (betaCMOOX) activity was not affected. Apo-12'carotenal and the retinoic acid receptor (RAR) alpha antagonist Ro 41-5253 significantly increased the intestinal enzyme activity by 55 and 94%, respectively. When beta-carotene was administered to rats pretreated with the two cytochrome P(450) (CYP) inducers, pentobarbital and naphthoflavone, the intestinal betaCMOOX activity increased by 39%. In a transcriptional study in chickens, treatment with retinoic acid resulted in low expression of the intestinal betaCMOOX. Our data suggest that retinoids and carotenoids might regulate betaCMOOX expression by a transcriptional feedback mechanism via interaction with members of the RAR family.