Apc(MIN) modulation of vitamin D secosteroid growth control

A central paradox of vitamin D biology is that 1alpha,25-(OH)(2) D(3) exposure inversely relates to colorectal cancer (CRC) risk despite a capacity for activation of both pro- and anti-oncogenic mediators including osteopontin (OPN)/CD44 and E-cadherin, respectively. Most sporadic CRCs arise from adenomatous polyposis coli (APC) gene mutation but understanding of its effects on vitamin D growth control is limited. Here we investigate effects of the Apc(Min/+) genotype on 1alpha,25-(OH)(2) D(3) regulation of OPN/CD44/E-cadherin signalling and intestinal tumourigenesis, in vivo. In untreated Apc(Min/+) versus Apc(+/+) intestines, expression levels of OPN and its CD44 receptor were increased, whereas E-cadherin tumour suppressor signalling was attenuated. Treatment by 1alpha,25-(OH)(2) D(3) or rationally designed analogues (QW or BTW) enhanced OPN but inhibited expression of CD44, the OPN receptor implicated in cell growth. These treatments also enhanced E-cadherin tumour suppressor activity, characterized by inhibition of beta-catenin nuclear localization, T-cell factor 1 and c-myelocytomatosis protein expression in Apc(Min/+) intestine. All secosteroids suppressed Apc(Min/+)-driven tumourigenesis although QW and BTW had lower calcium-related toxicity. Taken together, these data indicate that the Apc(Min/+) genotype modulates vitamin D secosteroid actions to promote functional predominance of E-cadherin tumour suppressor activity within antagonistic molecular networks. APC heterozygosity may promote favourable tissue- or tumour-specific conditions for growth control by vitamin D secosteroid treatment.

Effect of vitamin D on Epstein-Barr (EBV)-specific CD8+T cells in patients with early multiple sclerosis (MS)

Background: Infection with EBV and a lack in vitamin D may be important environmental triggers of MS. 1,25-(OH)2D3 mediates a shift of antigen presenting cells (APC) and CD4+ T cells to a less inflammatory profile. Although CD8+ T cells do express the vitamin D receptor, a direct effect of 1,25(OH)2D3 on these cells has not been demonstrated until now. Since CD8+ T cells are important immune mediators of the inflammatory response in MS, we examined whether vitamin D directly affects the CD8+ T cell response, and more specifically if it modulates the EBV-specific CD8+ T cell response. Material and Methods: To explore whether the vitamin D status may influence the pattern of the EBV-specific CD8+ T cell response, PBMC of 10 patients with early MS and 10 healthy controls (HC) were stimulated with a pool of immunodominant 8-10 mer peptide epitopes known to elicit CD8+ T cell responses. PBMC were stimulated with this EBV CD8 peptide pool, medium (negative control) or anti- CD3/anti-CD28 beads (positive control). The following assays were performed: ELISPOT to assess the secretion of IFN-gamma by T cells in general; cytometric beads array (CBA) and ELISA to determine whichcytokines were released by EBV-specific CD8+ T cells after six days of culture; and intracellular cytokine staining assay to determine by which subtype of T cells secreted given cytokines. To examine whether vitamin D could directly modulate CD8+ T cell immune responses, we depleted CD4+ T cells using negative selection. Results: We found that pre-treatment of vitamin D had an antiinflammatory action on both EBV-specific CD8+ T cells and on CD3/ CD28-stimulated T cells: secretion of pro-inflammatory cytokines (IFNgamma and TNF-alpha) was decreased, whereas secretion of antiinflammatory cytokines (IL-5 and TGF-beta) was increased. At baseline, CD8+ T cells of early MS patients showed a higher secretion of TNFalpha and lower secretion of IL-5. Addition of vitamin D did not restore the same levels of both cytokines as compared to HC. Vitamin D-pretreated CD8+T cells exhibited a decreased secretion of IFN-gamma and TNF-alpha, even after depletion of CD4+ T cells from culture. Conclusion: Vitamin D has a direct anti-inflammatory effect on CD8+ T cells independently from CD4+ T cells. CD8+ T cells of patients with earlyMS are less responsive to the inflammatory effect of vitamin D than HC, pointing toward an intrinsic dysregulation of CD8+ T cells. The modulation of EBV-specific CD8+T cells by vitaminDsuggests that there may be interplay between these twomajor environmental factors of MS. This study was supported by a grant from the Swiss National Foundation (PP00P3-124893), and by an unrestricted research grant from Bayer to RDP.

Benefits of Combinations of Vitamin A, C and E Derivatives in the Stability of Cosmetic Formulations

Chemically stable ester derivatives of vitamins A, C and E have become a focus of interest for their role in the satisfactory results in skin aging treatments. Accordingly, the aim of this study was to evaluate the physical and chemical stability of a cosmetic formulation containing 1% retinyl palmitate, ascorbyl tetraisopalmitate and tocopheryl acetate, alone or in combination. In the studies of physical stability, a Brookfield rheometer was used to determine rheological behavior of formulations containing the vitamins. Chemical stability was determined by HPLC on a Shimadzu system with UV detection. Results showed that formulations had pseudoplastic behavior and that vitamins did not alter their apparent viscosity and thixotropy. In the chemical stability studies, first-order reaction equations were used for determinations of the shelf-life of vitamins derivatives considering a remaining concentration of 85%. Combined vitamins in a single formulation had a slightly lower degradation rate as compared to different preparations containing only one of the vitamins. Considering that many cosmetic formulations contain vitamin combinations it is suggested that the present study may contribute to the development of more stable formulations containing liposoluble vitamins.

Retinoid -induced regression of human head and neck squamous cell carcinomas is associated with the induction of a pro -inflammatory response

Retinoids are Vitamin A derivatives that are effective chemopreventative and chemotherapeutic agents for head and neck squamous cell carcinomas (HNSCC). Despite the wide application of retinoids in cancer treatment, the mechanism by which retinoids inhibit head and neck squamous cell carcinomas is not completely understood. While in vitro models show that drugs affect cell proliferation and differentiation, in vivo models, such as tumor xenografts in nude mice drugs affect more complex parameters such as extracellular matrix formation, angiogenesis and inflammation. Therefore, we studied the effects of retinoids on the growth of the 22B HNSCC tumors using a xenograft model. In this system, retinoids had no effect on tumor cell differentiation but caused invasion of the tumor by inflammatory cells. Retinoid induced inflammation lead to tumor cell death and tumor regression. Therefore, we hypothesized that retinoids stimulated the 22B HNSCC xenografts to produce a pro-inflammatory signal such as chemokines that in turn activated host inflammatory responses. ^ We used real time quantitative RT-PCR to measure cytokine and chemokine expression in retinoid treated tumors. Treatment of tumors with an RAR-specific retinoid, LGD1550, had no effect on the expression of TNFα, IL-1α, GROα, IP-10, Rantes, MCP-1 and MIP-1α but induced IL-8 mRNA 5-fold. We further characterized the retinoid effect on IL-8 expression on the 22B HNSCC and 1483 HNSCC cells in vitro. Retinoids increased IL-8 expression and enhanced TNFα-dependent IL-8 induction. In addition, retinoids increased the basal and TNFα-dependent expression of MCP-1 but decreased the basal and TNFα dependent expression of IP-10. The effect of retinoids on IL-8 and MCP-1 expression was very rapid with increased levels of mRNA detected within 1–2 hours. This effect did not require new protein synthesis and did not result from mRNA stabilization. Both RAR and RXR ligands increased IL-8 expression whereas only RAR ligands activated MCP-1 expression. ^ We identified a functional retinoid response element in the IL-8 promoter that was located adjacent to the C/EBP-NFkB response element. TNFα treatment of the 22B cells caused rapid, transient and selective acetylation of regions of the IL-8 promoter associated with the NFkB response element. Co-treatment of the cells with retinoids plus TNF increased the acetylation of chromatin in this region without altering the kinetics of acetylation. These results demonstrate that ligand activated retinoid receptors can cooperate with NFkB in histone acetylation and chromatin remodeling. We believe that in certain HNSCC tumors this cooperation and the resulting enhancement of IL-8 expression can induce an inflammatory response that leads to tumor regression. We believe that the induction of inflammation in susceptible tumors, possibly coupled with cytotoxic interventions may be an important component in the use of retinoids to treat human squamous cancers. ^

Deoxycytidine glyoxal:Lesion induction and evidence of repair following vitamin C supplementation in vivo

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p = .001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p = .0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected. © 2003 Elsevier Science Inc.

Does a high UV environment ensure adequate vitamin D status?

This study assesses the Vitamin D status of 126 healthy free-living adults aged 18–87 years, in southeast Queensland, Australia (27°S) at the end of the 2006 winter. Participants provided blood samples for analysis of 25(OH)D (the measure of an individual’s Vitamin D status), PTH, Calcium, Phosphate, and Albumin, completed a questionnaire on sun-protective/sun-exposure behaviours, and were assessed for phenotypic characteristics such as skin/hair/eye colour and BMI. We found that 10.2% of the participants had serum 25(OH)D levels below 25 nmol/l (considered deficient) and a further 32.3% had levels between 25 nmol/l and 50 nmol/l (considered insufficient). Our results show that low levels of 25(OH)D can occur in a substantial proportion of the population at the end of winter, even in a sunny climate. 25(OH)D levels were higher amongst those who spent more time in the sun and lower among obese participants (BMI > 30) than those who were not obese (BMI < 30). 25(OH)D levels were also lower in participants who had black hair, dark/olive skin, or brown eyes, when compared with participants who had brown or fair hair, fair skin, or blue/green eyes. No associations were found between 25(OH)D status and age, gender, smoking status, or the use of sunscreen.

Investigating the links between muscle strength, sun exposure, dietary vitamin D intake and the vitamin D status of ambulatory older adults in South East Queensland

Vitamin D deficiency and insufficiency are now seen as a contemporary health problem in Australia with possible widespread health effects not limited to bone health1. Despite this, the Vitamin D status (measured as serum 25-hydroxyvitamin D (25(OH)D)) of ambulatory adults has been overlooked in this country. Serum 25(OH)D status is especially important among this group as studies have shown a link between Vitamin D and fall risk in older adults2. Limited data also exists on the contributions of sun exposure via ultraviolet radiation and dietary intake to serum 25(OH)D status in this population. The aims of this project were to assess the serum 25(OH)D status of a group of older ambulatory adults in South East Queensland, to assess the association between their serum 25(OH)D status and functional measures as possible indicators of fall risk, obtain data on the sources of Vitamin D in this population and assess whether this intake was related to serum 25(OH)D status and describe sun protection and exposure behaviors in this group and investigate whether a relationship existed between these and serum 25(OH)D status. The collection of this data assists in addressing key gaps identified in the literature with regard to this population group and their Vitamin D status in Australia. A representative convenience sample of participants (N=47) over 55 years of age was recruited for this cross-sectional, exploratory study which was undertaken in December 2007 in south-east Queensland (Brisbane and Sunshine coast). Participants were required to complete a sun exposure questionnaire in addition to a Calcium and Vitamin D food frequency questionnaire. Timed up and go and handgrip dynamometry tests were used to examine functional capacity. Serum 25(OH)D status and blood measures of Calcium, Phosphorus and Albumin were determined through blood tests. The Mean and Median serum 25-Hydroxyvitamin D (25(OH)D) for all participants in this study was 85.8nmol/L (Standard Deviation 29.7nmol/L) and 81.0nmol/L (Range 22-158nmol/L), respectively. Analysis at the bivariate level revealed a statistically significant relationship between serum 25(OH)D status and location, with participants living on the Sunshine Coast having a mean serum 25(OH)D status 21.3nmol/L higher than participants living in Brisbane (p=0.014). While at the descriptive level there was an apparent trend towards higher outdoor exposure and increasing levels of serum 25(OH)D, no statistically significant associations between the sun measures of outdoor exposure, sun protection behaviors and phenotypic characteristics and serum 25(OH)D status were observed. Intake of both Calcium and Vitamin D was low in this sample with sixty-eight (68%) of participants not meeting the Estimated Average Requirements (EAR) for Calcium (Median=771.0mg; Range=218.0-2616.0mg), while eighty-seven (87%) did not meet the Adequate Intake for Vitamin D (Median=4.46ug; Range=0.13-30.0ug). This raises the question of how realistic meeting the new Adequate Intakes for Vitamin D is, when there is such a low level of Vitamin D fortification in this country. However, participants meeting the Adequate Intake (AI) for Vitamin D were observed to have a significantly higher serum 25(OH)D status compared to those not meeting the AI for Vitamin D (p=0.036), showing that meeting the AI for Vitamin D may play a significant role in determining Vitamin D status in this population. By stratifying our data by categories of outdoor exposure time, a trend was observed between increased importance of Vitamin D dietary intake as a possible determinant of serum 25(OH)D status in participants with lower outdoor exposures. While a trend towards higher Timed Up and Go scores in participants with higher 25(OH) D status was seen, this was only significant for females (p=0.014). Handgrip strength showed statistically significant association with serum 25(OH)D status. The high serum 25(OH)D status in our sample almost certainly explains the limited relationship between functional measures and serum 25(OH)D. However, the observation of an association between slower Time Up and Go speeds, and lower serum 25(OH)D levels, even with a small sample size, is significant as slower Timed Up and Go speeds have been associated with increased fall risk in older adults3. Multivariable regression analysis revealed Location as the only significant determinant of serum 25(OH)D status at p=0.014, with trends (p=>0.1) for higher serum 25(OH)D being shown for participants that met the AI for Vitamin D and rated themselves as having a higher health status. The results of this exploratory study show that 93.6% of participants had adequate 25(OH)D status-possibly due to measurement being taken in the summer season and the convenience nature of the sample. However, many participants do not meet their dietary Calcium and Vitamin D requirements, which may indicate inadequate intake of these nutrients in older Australians and a higher risk of osteoporosis. The relationship between serum 25(OH)D and functional measures in this population also requires further study, especially in older adults displaying Vitamin D insufficiency or deficiency.

Investigating erythemal UV exposure and vitamin D production in the urban canyon

Exposure to ultraviolet radiation (UV) results in both damaging and beneficial health outcomes. Excessive UV exposure has been linked to many skin and eye problems, but moderate exposure induces vitamin D production. It has been reported that humans receive 90-95% of their vitamin D from production that starts after UV exposure. Although it is possible to acquire vitamin D through dietary supplementation, the average person receives very little in this manner. Therefore, since most people acquire their vitamin D from synthesis after exposure to UV from sunlight, it is very important to understand the different environments in which people encounter UV. This project measured UV radiation and in-vitro vitamin D production in the urban canyon and at a nearby suburban location. The urban canyon is an environment consisting of tall buildings and tropospheric air pollution, which have an attenuating effect on UV. Typically, UV measurements are collected in areas outside the urban canyon, meaning that at times studies and public recommendations do not accurately represent the amount of UV reaching street-level in highly urbanized areas. Understanding of UV exposure in urban canyons becomes increasingly important as the number of people working and living in large cities steadily increases worldwide. This study was conducted in the central business district (CBD) of Brisbane, Australia, which models the urban canyons of large cities around the world in that it boasts a great number of tall buildings, including many skyscrapers, meaning that most areas only see a small amount of direct sunlight each day. During the winter of 2007 measurements of UV radiation and in-vitro vitamin D production were collected in the CBD and at a suburban site approximately 2.5km outside the CBD. Air pollution data was obtained from a central CBD measurement site. Data analysis showed that urban canyon measurements of both UV radiation and in-vitro vitamin D production were significantly lower than those collected at the suburban site. These results will aid both future researchers and policy makers in better understanding human UV exposure in Brisbane’s CBD and other urban canyons around the world.

Willingness to trial functional foods and vitamin supplements: the role of attitudes, subjective norms, and dread of risks

Australian non-users of vitamin supplements (N = 162) and functional foods (N = 226) responded to a questionnaire examining their attitudes, subjective norms, and perceived behavioural control from the Theory of Planned Behaviour (TPB), risk dread and risk familiarity, and willingness to engage in free product trials. The impact of participants’ gender and age was also examined. Attitude and subjective norms were significant determinants of non-users willingness to trial each of the health products. Participants’ dread of the risk associated with the product was also a determinant of willingness to use functional foods. The overall models predicted between 25% and 30% of the variance in people’s willingness to trial the products. The findings provided some support for the TPB in predicting people’s willingness to trial functional foods and vitamin supplements and suggested, for willingness to trial functional foods, that non-users are also influenced by their dread of the risk associated with product use.

Vitamin D in health and disease : an insight into traditional functions and new roles for the 'sunshine vitamin'

Vitamin D is unique among the vitamins in that humans can synthesize it via the action of UV radiation upon the skin. This combined with its ability to act on specific target tissues via Vitamin D Receptor’s (VDR) make its classification as a steroid hormone more appropriate. While Vitamin D deficiency is a recognized problem in some northern latitude countries, recent studies have shown even in sunny countries such as Australia, vitamin D deficiency may be more prevalent than first thought. Vitamin D is most well known for its role in bone health, however, the discovery of VDR’s on a wide variety of tissue types has also opened up roles for vitamin D far beyond traditional bone health. These include possible associations with autoimmune diseases such as multiple sclerosis and inflammatory bowel diseases, cancer, cardiovascular diseases and muscle strength. Firstly, this paper presents an overview of the two sources of vitamin D: exposure to ultraviolet-B radiation and food sources of vitamin D, with particular focus on both Australian and international studies on dietary vitamin D intake and national fortification strategies. Secondly, the paper reviews recent epidemiological and experimental evidence linking vitamin D and its role in health and disease for the major conditions linked to suboptimal vitamin D, while identifying significant gaps in the research and possible future directions for research.

Vitamin D and sun protection : the impact of mixed public health messages in Australia

Exposure of the skin to sunlight can cause skin cancer and is also necessary for cutaneous vitamin D production. Media reports have highlighted the purported health benefits of vitamin D. Our aim was to examine attitudes and behaviours related to sun protection and vitamin D. A cross-sectional study of 2,001 residents in Queensland, Australia aged 20-70 years was undertaken. Information collected included: skin cancer risk factors; perceptions about levels of sun exposure required to maintain vitamin D; belief that sun protection increases risk of vitamin D deficiency; intention, and actual change in sun protection practices for adults and children. Multivariate models examined predictors of attitudinal and behavioural change. One-third (32%) believed a fair-skinned adult, and 31% thought a child required at least 30 minutes per day in summer sun to maintain vitamin D levels. Reductions in sun protection were reported by 21% of adults and 14% of children. Factors associated with belief that sun protection may result in not obtaining enough vitamin D included aged ≥ 60 years (OR=1.35, 95% CI 1.09-1.66) and having skin that tanned easily (OR=1.96, 95% CI 1.38-2.78). Participants from low income households, and those who frequently used sun protective clothing were more likely to have reduced sun protection practices (OR=1.33, 95% CI 1.10-1.73 and OR=1.73, 95% CI 1.36-2.20, respectively). This study provides evidence of reductions in sun protection practices in a population living in a high UV environment. There is an urgent need to re-focus messages regarding sun exposure and for continued sun protection practices.

Public health and clinical dilemmas resulting from imprecise vitamin D tests

Objective: To determine whether there are clinical and public health dilemmas resulting from the reproducibility of routine vitamin D assays. Methods: Blinded agreement studies were conducted in eight clinical laboratories using two commonly used assays to measure serum 25-hydroxyvitamin D (25(OH)D) levels in Australasia and Canada (DiaSorin Radioimmunoassay (RIA) and DiaSorin LIAISON® one). Results: Only one laboratory measured 25(OH)D with excellent precision. Replicate 25(OH)D measurements varied by up to 97% and 15% of paired results differed by more than 50%. Thirteen percent of subjects received one result indicating insufficiency [25-50 nmol/l] and another suggesting adequacy [>50 nmol/l]). Agreement ranged from poor to excellent for laboratories using the manual RIA, while the precision of the semi-automated Liaison® system was consistently poor. Conclusions: Recent interest in the relevance of vitamin D to human health has increased demand for 25(OH)D testing and associated costs. Our results suggest clinicians and public health authorities are making decisions about treatment or changes to public health policy based on imprecise data. Clinicians, researchers and policy makers should be made aware of the imprecision of current 25(OH)D testing so that they exercise caution when using these assays for clinical practice, and when interpreting the findings of epidemiological studies based on vitamin D levels measured using these assays. Development of a rapid, reproducible, accurate and robust assay should be a priority due to interest in populationbased screening programs and research to inform public health policy about the amount of sun exposure required for human health. In the interim, 25(OH)D results should routinely include a statement of measurement uncertainty.

Elucidating the links between UV radiation and vitamin D synthesis : using an in vitro model

Ultraviolet radiation (UV) is the carcinogen that causes the most common malignancy in humans – skin cancer. However, moderate UV exposure is essential for producing vitaminDin our skin. VitaminDincreases the absorption of calcium from the diet, and adequate calcium is necessary for the building and maintenance of bones. Thus, low levels of vitamin D can cause osteomalacia and rickets and contribute to osteoporosis. Emerging evidence also suggests vitamin D may protect against falls, internal cancers, psychiatric conditions, autoimmune diseases and cardiovascular diseases. Since the dominant source of vitamin D is sunlight exposure, there is a need to understand what is a “balanced” level of sun exposure to maintain an adequate level of vitamin D but minimise the risks of eye damage, skin damage and skin cancer resulting from excessive UV exposure. There are many steps in the pathway from incoming solar UV to the eventual vitamin D status of humans (measured as 25-hydroxyvitamin D in the blood), and our knowledge about many of these steps is currently incomplete. This project begins by investigating the levels of UV available for synthesising vitamin D, and how these levels vary across seasons, latitudes and times of the day. The thesis then covers experiments conducted with an in vitro model, which was developed to study several aspects of vitamin D synthesis. Results from the model suggest the relationship between UV dose and vitamin D is not linear. This is an important input into public health messages regarding ‘safe’ UV exposure: larger doses of UV, beyond a certain limit, may not continue to produce vitamin D; however, they will increase the risk of skin cancers and eye damage. The model also showed that, when given identical doses of UV, the amount of vitamin D produced was impacted by temperature. In humans, a temperature-dependent reaction must occur in the top layers of human skin, prior to vitamin D entering the bloodstream. The hypothesis will be raised that cooler temperatures (occurring in winter and at high latitudes) may reduce vitamin D production in humans. Finally, the model has also been used to study the wavelengths of UV thought to be responsible for producing vitamin D. It appears that vitamin D production is limited to a small range of UV wavelengths, which may be narrower than previously thought. Together, these results suggest that further research is needed into the ability of humans to synthesise vitamin D from sunlight. In particular, more information is needed about the dose-response relationship in humans and to investigate the proposed impact of temperature. Having an accurate action spectrum will also be essential for measuring the available levels of vitamin D-effective UV. As this research continues, it will contribute to the scientific evidence-base needed for devising a public health message that will balance the risks of excessive UV exposure with maintaining adequate vitamin D.

Environmental and pro-social norms : evidence on littering

The paper investigates the relationship between pro-social norms and its implications for improved environmentsl outcomes. This is an area, which has been neglected in the environmental economic literature. We provide empirical evidence to demonstrate a small but significant positive impact between perceived environmental cooperation (reduced public littering) and increased voluntary environmental morale. For this purpose we use European Value Survey (EVS) data for 30 European countries. We also demonstrate that Western European countries are more sensitive to perceived environmental cooperation than the public in Eastern Europe. Interestingly, the results also demonstrate that environmental morale is strongly correlated with several socio-economic and environmental variables. Several robustness tests are conducted to check the validity of the results.