Regina Goralczyk

CMO1 Deficiency Abolishes Vitamin A Production from β-Carotene and Alters Lipid Metabolism in Mice

Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency in humans. These plant-derived compounds must be cleaved and metabolically converted by intrinsic carotenoid oxygenases to support the panoply of vitamin A-dependent physiological processes. Two different carotenoid-cleaving enzymes were identified in mammals, the classical carotenoid-15,15′-oxygenase (CMO1) and a putative carotenoid-9′,10′-oxygenase (CMO2). To analyze the role of CMO1 in mammalian physiology, here we disrupted the corresponding gene by targeted homologous recombination in mice. On a diet providing β-carotene as major vitamin A precursor, vitamin A levels fell dramatically in several tissues examined. Instead, this mouse mutant accumulated the provitamin in large quantities (e.g. as seen by an orange coloring of adipose tissues). Besides impairments in β-carotene metabolism, CMO1 deficiency more generally interfered with lipid homeostasis. Even on a vitamin A-sufficient chow, CMO1-/- mice developed a fatty liver and displayed altered serum lipid levels with elevated serum unesterified fatty acids. Additionally, this mouse mutant was more susceptible to high fat diet-induced impairments in fatty acid metabolism. Quantitative reverse transcription-PCR analysis revealed that the expression of peroxisome proliferator-activated receptor γ-regulated marker genes related to adipogenesis was elevated in visceral adipose tissues. Thus, our study identifies CMO1 as the key enzyme for vitamin A production and provides evidence for a role of carotenoids as more general regulators of lipid metabolism.

Lycopene and vitamin E interfere with autocrine/paracrine loops in the dunning prostate cancer model

Epidemiological studies have consistently associated high intakes of lycopene or vitamin E with a reduced prostate cancer risk. Both compounds were tested in the MatLyLu Dunning prostate cancer model to gain insight into the in vivo action of lycopene and vitamin E. Supplementation for 4 weeks with 200 ppm lycopene, 540 ppm vitamin E, or both led to plasma levels comparable with those in humans. Both compounds also accumulated in tumor tissue. Macroscopic evaluation of the tumors by magnetic resonance imaging showed a significant increase in necrotic area in the vitamin E and the lycopene treatment groups. Microarray analysis of tumor tissues revealed that both compounds regulated local gene expression. Vitamin E reduced androgen signaling without affecting androgen metabolism. Lycopene interfered with local testosterone activation by down‐regulating 5‐α‐reductase and consequently reduced steroid target genes expression (cystatin‐related protein 1 and 2, prostatic spermine binding protein, prostatic steroid binding protein C1, C2 and C3 chain, probasin). In addition, lycopene down‐ regulated prostatic IGF‐I and IL‐6 expression. Based on these findings, we suggest that lycopene and vitamin E contribute to the reduction of prostate cancer by interfering with internal autocrine or paracrine loops of sex steroid hormone and growth factor activation/synthesis and signaling in the prostate.

Lycopene reduced gene expression of steroid targets and inflammatory markers in normal rat prostate

Epidemiological evidence links consumption of lycopene, the red carotenoid of tomato, to reduced prostate cancer risk. We investigated the effect of lycopene in normal prostate tissue to gain insight into the mechanisms, by which lycopene can contribute to primary prostate cancer prevention. We supplemented young rats with 200 ppm lycopene for up to 8 wk, measured the uptake into individual prostate lobes, and analyzed lycopene‐induced gene regulations in dorsal and lateral lobes after 8 wk of supplementation. Lycopene accumulated in all four prostate lobes over time, with all‐trans lycopene being the predominant isoform. The lateral lobe showed a significantly higher total lycopene content than the other prostate lobes. Transcriptomics analysis revealed that lycopene treatment mildly but significantly reduced gene expression of androgen‐metabolizing enzymes and androgen targets. Moreover, local expression of IGF‐I was decreased in the lateral lobe. Lycopene also consistently reduced transcript levels of proinflammatory cytokines, immunoglobulins, and immunoglobulin receptors in the lateral lobe. This indicates that lycopene reduced inflammatory signals in the lateral prostate lobe. In summary, we show for the first time that lycopene reduced local prostatic androgen signaling, IGF‐I expression, and basal inflammatory signals in normal prostate tissue. All of these mechanisms can contribute to the epidemiologically observed prostate cancer risk reduction by lycopene.

β-carotene and lung cancer in smokers: review of hypotheses and status of research.

A number of epidemiological studies have reported associations of β-carotene plasma levels or intake with decreased lung cancer risk. However, intervention studies in smokers have unexpectedly reported increased lung tumor rates after high, long-term, β-carotene supplementation. Recently, detailed analyses by stratification for smoking habits of several large, long-term intervention or epidemiological trials are now available. The ATBC study, the CARET study, the Antioxidant Polyp Prevention trial, and the E3N study provide evidence that the adverse effects of β-carotene supplementation are correlated with the smoking status of the study participants. In contrast, the Physician Health Study, the Linxian trial, and a pooled analysis of 7 epidemiological cohort studies have not supported this evidence. The ferret and A/J mouse lung cancer model have been used to investigate the mechanism of interaction of β-carotene with carcinogens in the lung. Both models have specific advantages and disadvantages. There are a number of hypotheses concerning the β-carotene/tobacco smoke interaction including alterations of retinoid metabolism and signaling pathways and interaction with CYP enzymes and pro-oxidation/DNA oxidation. The animal models consistently demonstrate negative effects only in the ferret, and following dosing with β-carotene in corn oil at pharmacological dosages. No effects or even protective effects against smoke or carcinogen exposure were observed when β-carotene was applied at physiological dosages or in combination with vitamins C and E, either as a mixture or in a stable formulation. In conclusion, human and animal studies have shown that specific circumstances, among them heavy smoking, seem to influence the effect of high β-carotene intakes. In normal, healthy, nonsmoking populations, there is evidence of beneficial effects.

Dietary constituents reduce lipid accumulation in murine C3H10 T1/2 adipocytes: A novel fluorescent method to quantify fat droplets

BackgroundAdipocyte volume (fat accumulation) and cell number (adipogenesis) is increased in obese individuals. Our objective was the identification of dietary constituents with inhibitory effects on triglyceride formation during adipogenesis. Therefore an in vitro adipose cell assay in murine C3H10 T1/2 cells was developed, which enabled rapid quantification of intracellular fat droplet accumulation during adipocyte differentiation. Results were corroborated by expression levels of several specific adipogenic and lipogenic genes which are known to regulate triglyceride accumulation.MethodsC3H10 T1/2 adipocyte differentiation was conducted with rosiglitazone in the presence of test compounds for 7 days. Accumulation of intracellular lipid droplets was measured using the Cellomics® ArrayScan® VTI HCS reader and SpotDetector® BioApplication from ThermoFisher. Fluorescent images were automatically acquired and analysed employing the fluorescent dyes BODIPY® 493/503 and Hoechst 33342, for staining neutral lipids and localisation of nuclei, respectively. The expression levels of adipogenic and lipogenic genes, such as PPARα and PPARγ, C/EBPα, aP2, adiponectin, LPL and HSL, CPT-1β, ACC1, Glut4 and FAS, were determined by quantitative RT-PCR. Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation.ResultsThe ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation. Additionally, it was demonstrated that adipogenic and lipogenic gene expression was attenuated. DHA, β-carotene and hydroxytyrosol inhibited the gene expression of PPARγ, C/EBPα, aP2 and CPT-1β.ConclusionThis in vitro assay in differentiating adipocytes enables automated detection and quantification of changes in lipid droplet number, size and intensity. The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and β-carotene correlate with the modulation of genes involved in adipocyte differentiation.

Monoamine reuptake inhibition and mood-enhancing potential of a specified oregano extract

A healthy, balanced diet is essential for both physical and mental well-being. Such a diet must include an adequate intake of micronutrients, essential fatty acids, amino acids and antioxidants. The monoamine neurotransmitters, serotonin, dopamine and noradrenaline, are derived from dietary amino acids and are involved in the modulation of mood, anxiety, cognition, sleep regulation and appetite. The capacity of nutritional interventions to elevate brain monoamine concentrations and, as a consequence, with the potential for mood enhancement, has not been extensively evaluated. The present study investigated an extract from oregano leaves, with a specified range of active constituents, identified via an unbiased, high-throughput screening programme. The oregano extract was demonstrated to inhibit the reuptake and degradation of the monoamine neurotransmitters in a dose-dependent manner, and microdialysis experiments in rats revealed an elevation of extracellular serotonin levels in the brain. Furthermore, following administration of oregano extract, behavioural responses were observed in mice that parallel the beneficial effects exhibited by monoamine-enhancing compounds when used in human subjects. In conclusion, these data show that an extract prepared from leaves of oregano, a major constituent of the Mediterranean diet, is brain-active, with moderate triple reuptake inhibitory activity, and exhibits positive behavioural effects in animal models. We postulate that such an extract may be effective in enhancing mental well-being in humans.

Skin Photoprotection by Carotenoids

In Western populations, a lifestyle favouring tanned skin leads to increased exposure to natural and artificial sources of UV-radiation (UVR). To keep the adverse effects of this exposure, such as sunburn, immunosuppression, photoaging and photocarcinogenesis, to a minimum, nutritional manipulation of the basic endogenous protective properties of skin is an attractive target. In this respect, considerable interest has been directed for many years towards the dietary carotenoids, because of their radical scavenging and singlet oxygen quenching properties and thus their putative role in photochemistry, photobiology and photomedicine.

β-Carotene-Induced Changes in RARβ Isoform mRNA Expression Patterns Do Not Influence Lung Adenoma Multiplicity in the NNK-Initiated A/J Mouse Model

Abstract: A number of epidemiological studies have reported associations of β-carotene plasma levels or intake with decreased lung cancer risk. However, intervention studies in smokers reported increased lung tumor rates after high long-term β-carotene supplementation. For insight into these conflicting results, we studied the influence of β-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 RARβ 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 β-carotene levels of up to 3 μmol/L within 4 wk and up to 6 μmol/L after 6 mo of supplementation on a diet modified to enhance β-carotene absorption. Despite high lung β-carotene concentrations of up to 6 μmol/kg, tumor multiplicity was not significantly affected by the β-carotene treatment, either in carcinogen-initiated or non-initiated mice, and was unrelated to β-carotene dose and the time point of treatment during cancer formation. Tumor multiplicity did not correlate with β-carotene plasma levels in NNK-treated animals. All RARβ isoforms were significantly suppressed in the lungs of NNK- and NNK plus high dose β-carotene-treated animals. However, the number of tumors per mouse did not correlate with the RARβ-isoform expression levels. β-carotene alone after 3 mo of supplementation mildly but significantly increased levels of RARβ1, β2, and β4. This increase persisted for 6 mo for RARβ2 and β4. In summary, we found no effect of β-carotene on tumor formation in the NNK-initiated A/J-mouse lung cancer model with respect to dose or time point of treatment. β-Carotene-induced changes in RARβ isoform gene expression levels were not predictive for the number of lung tumors but were indicative of intact β-carotene metabolism and persistent sensitivity to retinoic acid in the mice. Down-regulation of RARβ 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.

β-Carotene interference with UVA-induced gene expression by multiple pathways

UVA exposure causes skin photoaging by singlet oxygen (1O2)-mediated induction of matrix metalloproteases (MMPs). We assessed whether β-carotene, a carotenoid known as 1O2 quencher and retinoic acid (RA) precursor, interferes with UVA-induced gene regulation and prevents UVA-induced gene regulation in HaCaT human keratinocytes. HaCaT cells accumulated β-carotene in a time- and dose-dependent manner. UVA irradiation massively reduced the cellular β-carotene contents. β-Carotene suppressed UVA induction of MMP-1, MMP-3, and MMP-10 - three major MMPs involved in photoaging. HaCaT cells produced weak retinoid activity from β-carotene, as demonstrated by mild up-regulation of retinoid receptor RARβ and activation of an RARE-dependent reporter gene. Of the 568 UVA-regulated genes, β-carotene reduced the UVA effect for 143, enhanced it for 180, and did not interact with UVA for 245 genes. The pathways regulated β-carotene in interaction with UVA were characterized by genes involved in growth factor signaling, stress response, apoptosis, cell cycle, extracellular matrix (ECM) degradation, tanning, and inflammation. In conclusion, β-carotene at physiological concentrations interacted with UVA effects by multiple mechanisms that included, but were not restricted to, 1O2 quenching. With our results, we provide a mechanistic basis for the long-known and clinically established photoprotective effects of β-carotene in human skin.