Optimal serum calcidiol concentration for cancer prevention.

UNLABELLED: It has been demonstrated in several studies that serum calcidiol (25 OH vitamin D(3)) concentration is in a reversed and linear relationship with cancer risk. However, there are also studies showing no such association and some even suggest the opposite. The risk of pancreatic and oesophageal cancer seems to increase, when serum calcidiol concentration increases. A bias in these studies might be that their basic assumption is linear dependence of cancer on serum calcidiol concentration. Some studies suggest a U-shaped association between the disease and the serum calcidiol concentration. Evidence, in the literature, of the relationship between serum calcidiol concentration and disease is reviewed and an optimal level of 40-80 nmol/L (16-32 ng/ml) is suggested. Serum calcidiol seems to be a better predictor of cancer development than calcitriol (1α, 25 (OH)(2) vitamin D(3)). A calcidiol insufficiency, as well as an insufficient solar exposure, is associated with an increased risk of several solid carcinomas. In a recent study, our group demonstrated that calcidiol is an active hormone in CYP24 (24-hydroxylase) deficient cells. In these cells, calcidiol and calcitriol act synergistically, therefore fluctuations of the serum calcidiol concentration may define the hormonal activity and cancer development. CONCLUSION: Serum calcidiol concentration and the risk of many common diseases and aging phenomena seem to show a U-shaped association suggesting a lower and upper limit for healthy serum calcidiol concentration. An imbalance of hormonal calcidiol rather than that of calcitriol is a risk factor in carcinomas and chronic diseases, which might be prevented by an optimal serum calcidiol concentration. Multiple daily dosing of cholecalcipherol or skin patches could best provide an optimal dosing and stable serum concentration. Alternatively, narrow-band UV-B lamps are a possible optimal solution, when given by trained personnel.

Niacin in the prevention of atherosclerosis: Significance of vasodilatation.

There is a rising interest towards the old drug, nicotinic acid (niacin, vitamin B(3)), because at pharmacological concentrations it has a beneficial effect on HDL cholesterol. Its use, however, was limited due to its adverse effect, flushing. When the mechanism of flushing was solved, a combination of niacin and DP1 receptor antagonist or prostaglandin inhibitor is used, there has been a comeback of niacin with extensive clinical trials. This paper argues that the new strategy with niacin for the prevention of atherosclerosis should be re-evaluated, because vasodilatation of the peripheral vessels might be crucially important in the early primary prevention according to our "vasa vasorum hypoxia" hypothesis.

Narrowband ultraviolet B course improves vitamin D balance in women in winter.

BACKGROUND: Vitamin D insufficiency is common in winter in the Nordic countries. OBJECTIVES: To examine whether a short course of narrowband ultraviolet B (NB-UVB) improves vitamin D balance. METHODS: Fifty-six healthy, white women (mean age 41 years) volunteered and 53 completed the study. NB-UVB exposures were given on seven consecutive days either on the whole body (n = 19), on the head and arms (n = 9) or on the abdomen (n = 14). Similarly, seven solar simulator exposures were given on the face and arms (n = 11). The cumulative UVB dose was 13 standard erythema doses in all regimens. Serum calcidiol (25-hydroxyvitamin D) concentration was measured by radioimmunoassay before and after the NB-UVB exposures. Follow-up samples were taken from the whole-body NB-UVB group at 2 months. RESULTS: At onset 41 women (77%) had vitamin D insufficiency (calcidiol < 50 nmol L(-1)) and six (11%) had vitamin D deficiency (calcidiol < 25 nmol L(-1)). Calcidiol concentration increased significantly, by a mean of 11.4 nmol L(-1) when NB-UVB was given on the whole body, by 11.0 nmol L(-1) when given on the head and arms and by 4.0 nmol L(-1) when given on the abdomen. Solar simulator exposures given on the face and arms increased calcidiol by 3.8 nmol L(-1). After 2 months serum calcidiol was still higher than initially in the group who received NB-UVB exposures on the whole body. CONCLUSIONS: NB-UVB exposures given on seven consecutive days on different skin areas of healthy women significantly improved serum calcidiol concentration. A short low-dose NB-UVB course can improve vitamin D balance in winter.

Vitamin D, nervous system and aging.

This is a mini-review of vitamin D(3), its active metabolites and their functioning in the central nervous system (CNS), especially in relation to nervous system pathologies and aging. The vitamin D(3) endocrine system consists of 3 active calcipherol hormones: calcidiol (25OHD(3)), 1alpha-calcitriol (1alpha,25(OH)2D(3)) and 24-calcitriol (24,25(OH)2D(3)). The impact of the calcipherol hormone system on aging, health and disease is discussed. Low serum calcidiol concentrations are associated with an increased risk of several chronic diseases including osteoporosis, cancer, diabetes, autoimmune disorders, hypertension, atherosclerosis and muscle weakness all of which can be considered aging-related diseases. The relationship of many of these diseases and aging-related changes in physiology show a U-shaped response curve to serum calcidiol concentrations. Clinical data suggest that vitamin D(3) insufficiency is associated with an increased risk of several CNS diseases, including multiple sclerosis, Alzheimer's and Parkinson's disease, seasonal affective disorder and schizophrenia. In line with this, recent animal and human studies suggest that vitamin D insufficiency is associated with abnormal development and functioning of the CNS. Overall, imbalances in the calcipherol system appear to cause abnormal function, including premature aging, of the CNS.

Heliotherapy improves vitamin D balance and atopic dermatitis.

BACKGROUND: Vitamin D insufficiency during winter is common in the Nordic countries. Heliotherapy (HT) may heal atopic dermatitis (AD) but its effect on vitamin D balance has not been examined. OBJECTIVES: To study the effect of HT on serum calcidiol (25-hydroxyvitamin D) concentration and on healing of AD. METHODS: Twenty-three adult patients with AD received a 2-week course of HT in the Canary Islands in either January or March 2005. Daily solar ultraviolet (UV) radiation was measured and personal UV exposure calculated as standard erythema doses (SED). Blood samples were taken during HT and during a 1-2 month follow-up. Serum calcidiol concentration was measured by radioimmunoassay. Healing of AD was examined by SCORAD index. RESULTS: Before HT 17 (74%) AD patients had vitamin D insufficiency (calcidiol < 50 nmol L(-1)) and four patients high (> 80 nmol L(-1)) serum calcidiol values. The median personal UV dose during the 2-week HT course was 60 SED in the January group and 109 SED in the March group. Serum calcidiol concentration increased significantly in both groups, by 13.4 and 24.0 nmol/L(-1), respectively, and after HT only four (17%) patients had vitamin D insufficiency. SCORAD improved from 34 to 9 in the January HT group and from 30 to 9 in the March group. CONCLUSIONS: A 2-week course of HT significantly improved vitamin D balance by increasing serum calcidiol concentration, and caused a marked healing of AD. These parallel positive responses should be taken into account when the benefits of HT are considered.

Involvement of nuclear progesterone receptors in the formation of anxiety in female mice.

We report here studies on the delayed effects of exogenous progesterone on the formation of anxiety in female mice. Ovariectomized female mice were given seven days of replacement therapy either with the two main ovarian hormones-progesterone and estradiol benzoate-or with progesterone only; levels of anxiety were measured six hours later in the elevated plus-maze. The role of nuclear progesterone receptors in controlling the level of anxiety was assessed by giving some mice injections of the synthetic progesterone receptor blocker mifepristone 2 h before the last dose of hormones. An immunohistochemical method was used to study changes in the number of nuclear progesterone receptors in different areas of the brains of experimental animals. These studies showed that progesterone has a delayed enhancing effect on anxiety in female mice. The role of nuclear progesterone receptors in forming this behavioral characteristic was supported by a strong correlation between changes in the numbers of progesterone receptor-immunopositive cells in several brain structures and the level of anxiety. Prior blockade of progesterone receptors using mifepristone led to a maximal reduction in the level of anxiety, which was also evidence for a role for the genomic mechanisms of action of progesterone in controlling anxiety in females.

Pharmacological modulation of anxiety-related behaviors in the murine Suok test.

We have recently introduced a new model of anxiety--the Suok test and its light-dark modification--for behavioral characterization in mice and rats, including simultaneous assessment of their anxiety, activity, and neurological phenotypes. In the present study, testing different inbred (129S1, BALB/c) and hybrid (C57-129S1) mouse strains in both Suok test modifications, we examined the effects on anxiety-related behaviours produced by traditional anxiogenic and anxiolytic drugs. Here we show dose-dependent increases in anxiety-related behaviors produced by anxiogenic drug pentylenetetrazole (10 and 20 mg/kg). In contrast, anxiolytic drugs ethanol (0.75 and 1.5 g/kg) and diazepam (0.5 mg/kg) reduced anxiety and increased mouse exploration in this test. Hyperemotional anxious BALB/c mice were particularly sensitive to pharmacogenic anxiety in Suok test, also showing robust light-dark shifts in the light-dark version of this test. Overall, the results of this study confirm the potential utility of both murine Suok tests, especially when used in selected "sensitive" mouse strains, for high-throughput screening of potential anxiotropic drugs.

The vitamin D neuroendocrine system as a target for novel neurotropic drugs.

Vitamin D is a seco-steroid hormone with multiple functions in the nervous system. Physiological brain mechanisms of vitamin D and its receptors include neuroprotection, antiepileptic effects, immunomodulation, possible interplay with several brain neurotransmitter systems and hormones, as well as the regulation of behaviours. Here we review the important role of the vitamin D neuroendocrine system in the brain, and outline perspectives for the search for novel neurotropic drugs to treat various vitamin D-related dysfunctions.

Altered levels of Smad2 and Smad4 are associated with human prostate carcinogenesis.

Alterations have been demonstrated in ligand and cognate receptor system of the transforming growth factor beta (TGF-beta) pathway in prostate cancer (PC). Still, little is known about changes in the activity of the intracellular Smad cascade of TGF-beta signaling during prostate carcinogenesis. We used immunohistochemistry to analyze phosphorylated Smad2 (p-Smad2), nuclear Smad4 and inhibitory-Smad7 in epithelial cells of normal, hyperplastic and malignant prostate. Specimens comprised 49 tissue cores of PC, 10 benign prostate hypertrophies and three normal prostates. Nuclear p-Smad2 (P<0.001) and nuclear Smad4 (P=0.023) were significantly decreased in PC with remarkable variations in cytoplasmic Smad7 levels. Substantial decreases in p-Smad2 and Smad4 levels were found in specimens with primary Gleason grades 3 and 4, whereas in grade 5, levels were markedly higher. Our results provide the first evidence for changes and reversible attenuation in the Smad system of the TGF-beta pathway during prostate carcinogenesis.

Vitamin D fortification as public health policy: significant improvement in vitamin D status in young Finnish men.

BACKGROUND: Vitamin D insufficiency is common in northern countries during wintertime. In Finland, after the recommendation by the Ministry of Social Affairs and Health, vitamin D has been added to liquid milk products and margarines from February 2003. OBJECTIVE: We determined the effects of national policy on vitamin D fortification on vitamin D status among young Finnish men. DESIGN: A comparison before and after intervention with study population of 196 young Finnish men (18-28 years) was carried out. Serum 25-hydroxyvitamin D3 (25-OHD3) concentrations were determined with the OCTEIA enzymeimmunoassay by IDS (Immunodiagnostic Systems Limited, Bolden, UK) in January 2003 (n = 96) and in January 2004 (n = 100), nearly 1 year after national vitamin D fortification had started. RESULTS: The mean serum 25-OHD3 concentrations during the wintertime increased by 50% after implementation of the vitamin D fortification of dairy products. Correspondingly, the prevalence of vitamin D insufficiency (serum 25-OHD3 < 40 nmol/l) was decreased by 50% from 78% in January 2003 to 35% in January 2004. CONCLUSIONS: Our results demonstrate that national vitamin D fortification substantially improved the vitamin D status of young Finnish men. Still, a third remained vitamin D insufficient.

Hair barbering in mice: implications for neurobehavioural research.

Barbering (fur/whisker trimming, the Dalila effect) is a behaviour-associated hair and whisker loss frequently seen in laboratory rodents, including mice. Here we analyse barbering behaviour in 129S1, NMRI, C57BL/6 and BALB/c mouse strains and some of their F1 hybrids. Our study shows that barbering in mice, depending on their genotype, is a complex behaviour with several distinct contexts or domains. We observed social (dominant) barbering in NMRI and C57BL/6 mice, sexual over-grooming in 129S1 and C57BL/6 mice, maternal barbering in lactating 129S1 and C57BL/6 mice, and stress-evoked barbering in F1 (NMRIx129S1) hybrids. In contrast, aggressive BALB/c mice and their F1 progeny do not use barbering in their behaviour. We suggest that barbering may be an important complex multi-domain behaviour sensitive to various manipulations, and represent a useful index in neurobehavioural research.

[Participation of progesterone receptors in plus-maze behavior in female mice].

The current study tested delayed effect pf progesterone on the anxiety level of female mice. The elevated plus maze (EPM) behavior was assessed in ovariectomized mice injected for 7 days with estradiol benzoate and progesterone or progesterone alone after 6 hrs of the last treatment. One group of ovariectomized mice was injected with progesterone receptor blocker Mifepristone before 2 hrs of the last treatment. The immunocytochemistry method was used to visualize cells in different brain areas having immunoreactivity (ir) for progesterone receptors. In the EPM, progesterone administration significantly increased the anxiety levels of ovariectomized mice as compared with estradiol benzoate and progesterone administration. The participation of nuclear progesterone receptors in anxiety levels regulation is confirmed by high correlation of the change of progesterone receptor-ir cell number in some brain areas and anxiety levels. Mifepristone decreased anxiety levels and progesterone receptor-ir cell number in both groups of mice that suggests involvement of genomic mechanisms in anxiety regulation in female mice.

The role of hair in swimming of laboratory mice: implications for behavioural studies in animals with abnormal hair.

Animal swimming tests, such as the forced swim test, are extensively used in biomedical research to study rodent behaviour. Hair and skin exposed to water may be an important factor affecting the performance in this test. Since various hair and skin abnormalities are not uncommon in genetically modified or drug-treated laboratory animals, this test may be inappropriate for these animals. Because on occasions it is necessary to screen their swimming behaviour, in the present study we aimed to assess the role of hair in swimming of laboratory rodents in the forced swim test, widely used in behavioural research. For this, we shaved laboratory mice (129S1 strain) and compared their swimming patterns with those of unshaven controls. Overall, shaving mice did not affect their swimming behaviours in the 5 min forced swim test. Our results indicate that hair condition is not an important factor in the forced swim test for this mouse strain, and suggest that this test may have wider utility for behavioural analyses of mice with abnormal hair.

Calcidiol and prostate cancer.

Epidemiological studies suggest that serum calcidiol (25(OH)-Vitamin D3) seems to be associated with several cancers including prostate cancer. We have made several experimental studies in order to clarify the mechanism(s) involved in the association. Calcidiol has been regarded as an inactive prohormone for calcitriol, which possesses the highest biological activity of the Vitamin D metabolites, when it is evaluated on the basis of bioactivity/nmol. However, we found recently that at the physiological concentration calcidiol (100-200 nM) is an active hormone, whereas calcitriol (1alpha,25(OH)2-Vitamin D3) (100 pM) is inactive in human primary prostate stromal cells. Calcidiol is able to inhibit cell growth and to induce or inhibit several genes including 1alpha-hydroxylase and 24-hydroxylase genes. This suggests that calcidiol might be an independent endocrine system involved in the control of cell differentiation and proliferation, whereas calcitriol might be mainly involved in the regulation of calcium and phosphorous balance. Several mechanisms may mediate the action of Vitamin D in the prostate. This is a review of some recent studies on the role of (1) Vitamin D metabolism, (2) growth factors and (3) fatty acid metabolism.

Small stable RNA level depends on the physiological state of the cell.

The level of 5S rRNA and tRNAi(Met)1 synthesized by RNA polymerase III was investigated in human epidermoid carcinoma cells A431 at different physiological states: low and high proliferation and apoptosis. The real-time RT-PCR method using SYBR Green I was applied to measure certain RNA species in total cellular RNA. The share of 5S rRNA was practically the same in slowly and actively proliferating A431 cells, but increased about 2.5-fold in apoptotic cells. The share of initiator tRNAi(Met)1 in actively proliferating and apoptotic cells was 1.5-2.0 times higher than in slowly proliferating cells. Our results suggest a possible existence of special mechanisms regulating RNA polymerase III-directed transcription from different type promoters in accordance with the physiological state of the cell.

Mechanisms of neuroprotective action of vitamin D(3).

This review considers modern data on the mechanisms underlying the neuroprotective effect of the neurosteroid vitamin D(3) and its receptors in the nervous system. Special attention is paid to Ca2+ regulation, stimulation of neurotrophin release, interaction with reactive oxygen and nitrogen species, and neuroimmunomodulatory effects of calcitriol, the main biologically active form of vitamin D(3), in the nervous system.

Increased grooming behavior in mice lacking vitamin D receptors.

Vitamin D is a neuroactive secosteroid with several important functions in the nervous system. Many human and animal findings link alterations in the vitamin D system to various neurological and behavioral disorders. Since grooming is an important element of animal behavior, here we studied whether genetic ablation of vitamin D receptors (VDR) in mice may be associated with altered grooming behaviors. Overall, VDR knockout (VDRko) mice presented longer duration and higher frequency of grooming when tested in the actimeter, open field, elevated plus maze, and horizontal rod tests. Increased grooming did not, however, correlate with unaltered general activity level (actimeter test), anxiety-like behaviors (hole board and elevated plus maze tests), and emotional reactivity index (defecation boli). In general, our results confirm the role of vitamin D and VDR in the regulation of behavior, including grooming, and suggest that increased grooming behavioral phenotype may be associated with genetic ablation of VDR in mutant mice.

The role of Vitamin D3 metabolism in prostate cancer.

Vitamin D deficiency increases risk of prostate cancer. According to our recent results, the key Vitamin D hormone involved in the regulation of cell proliferation in prostate is 25(OH) Vitamin D3. It is mainly acting directly through the Vitamin D receptor (VDR), but partially also through its 1alpha-hydroxylation in the prostate. A deficiency of 25(OH) Vitamin D is common especially during the winter season in the Northern and Southern latitudes due to an insufficient sun exposure, but Vitamin D deficient diet may partially contribute to it. A lack of Vitamin D action may also be due to an altered metabolism or Vitamin D resistance. Vitamin D resistance might be brought up by several mechanisms: Firstly, an increased 24-hydroxylation may increase the inactivation of hormonal Vitamin D metabolites resulting in a Vitamin D resistance. This is obvious in the cancers in which an oncogenic amplification of 24-hydroxykase gene takes place, although an amplification of this gene in prostate cancer has not yet been described. During the aging, the activity of 24-hydroxylase increases, whereas 1alpha-hydroxylation decreases. Furthermore, it is possible that a high serum concentration of 25(OH)D3 could induce 24-hydroxylase expression in prostate. Secondly, Vitamin D receptor gene polymorphism or defects may result in a partial or complete Vitamin D resistance. Thirdly, an overexpression or hyperphosphorylation of retinoblastoma protein may result in an inefficient mitotic control by Vitamin D. Fourthly, endogenous steroids (reviewed by [D.M. Peehl, D. Feldman, Interaction of nuclear receptor ligands with the Vitamin D signaling pathway in prostate cancer, J. Steroid Biochem. Mol. Biol. (2004)]) and phytoestrogens may modulate the expression of Vitamin D metabolizing enzymes. In summary, the local metabolism of hormonal Vitamin D seems to play an important role in the development and progression of prostate cancer.

[Use of the real-time RT-PCR method for investigation of small stable RNA expression level in human epidermoid carcinoma cells A431]. / Ispol'zovanie metoda RT-PCR v real'nom vremeni dlia izucheniia urovnia ékspressii malykh stabil'nykh RNK v kletkakh épidermoidnoi kartsinomy cheloveka A431.

Real-time RT-PCR using fluorescence dyes (e.g. SYBR Green I) is currently the most sensitive and precise method for investigation of RNA level and has long been widely used for absolute and relative quantification of mRNA in the cell. This highly sensitive method allows measurement of different type RNA level in the cell based on the kinetics of the corresponding double-stranded cDNA amplification. Upon its binding to the minor groove of double-stranded DNA, SYBR Green I dye increases its fluorescence about 100-fold, and this increase can be recorded even at early cycles of amplification. During the real-time RT-PCR procedure the level of amplified DNA is measured after every cycle of amplification, which permits to perform quantification at the cycles when amplification curve has not yet reached the "plateau" range and corresponds to the range of exponential increase in DNA amount. This approach makes it possible to avoid misinterpretation of data typical of conventional PCR methods "in the end point" and caused by a deficiency of one or more reaction components at the late PCR cycles. We applied for the first time real-time RT-PCR using SYBR Green I for the measurement of the class III genes RNA-product level, that is, small stable non-translated RNAs--ribosomal 5S rRNA, initiator transfer RNAiMet1, and Alu-RNA, synthesized by DNA-dependent RNA polymerase III. We investigated the level of 5S rRNA-, tRNA- and Alu-gene expression in the cell being in different states: with prolonged generation period, activated to proliferation, and apoptotic. The expression level was judged from the content of corresponding RNA-products in the total cellular RNA. The used approach enabled us to find out the specific RNA share in the total cell RNA. Human epidermoid carcinoma cells A431 were used as a model for investigating class III gene expression level in vivo. These cells expose on their surface an abnormally large amount of receptors to epidermoid growth factor (EGF), and the result of EGF action on A431 cells depends on the growth factor concentration. Low concentrations of EGF (0.1 ng/ml) cause active proliferation of A431 cells, but its high concentrations (10-100 ng/ml) cause apoptosis in these cells. Besides, upon growing in serum-free media, A431 cells continue to proliferate, but by this extending the generation period to 48 h, against 30 h on growing in serum-containing media. Hence, A431 cells can serve as a useful model for investigation of specific gene expression level in cells being in different physiological states, in both slowly and actively proliferating cells, and in apoptotic cells. For successful use of real-time RT-PCR in 5S rRNA, tRNAi(Met)1 and Alu-RNA level quantification, we optimized the amplification reaction conditions. We took into account that the share of each particular RNA in the cell may vary--the share of ribosomal RNA is high, tRNAi(Met)1--low, and Alu-RNA--very low. Moreover, the level of some small RNAs (e.g. Alu-RNA) can vary significantly in cells of different lines. This explains why the amount of cDNA, gained by reverse transcription of total cellular RNA, and the concentration of specific primers used for PCR were different in each case. We showed that the expression of different class III genes--5S rRNA-, tRNA- and Alu-genes, was not similarly regulated in response to external stimuli, causing prolongation of generation period, activation of proliferation and apoptosis. 5S rRNA level was practically the same in A431 cells both having prolonged generation period and being activated by EGF in low concentration, but in apoptotic cells this level dramatically fell about 8-fold. Alu-RNA level was equal in cells with prolonged generation period and in apoptotic cells, and increased about 2-fold in cells activated by EGF in low concentration. The initiator tRNAi(Met)1 level in cells activated by EGF in low concentration and in apoptotic cells was by almost two times higher than in cells with prolonged generation period. The data obtained testify that the real-time RT-PCR method using SYBR Green I yields highly reliable and reproducible quantification for the level of class III gene RNA-products--small stable RNAs (5S rRNA, tRNA and Alu-RNA). Examination of each specific RNA level requires individual selection for the amplification reaction conditions: the amount of cDNA and primer concentration in the sample. This is primarily caused by different expression levels in some particular class III genes within the frames of the cells, and by different levels of some small stable RNAs (e. g. Alu-RNA) in different cell lines. Special attention must be paid to the internal control for discriminating between specific RNA levels in proliferating and apoptotic cells, as in the late apoptosis RNAs of most types are degraded (for example, mRNA of "house-keeping" gene for RPLP0 protein, used as a possible internal control in our experiments). As far as the applied approach allows estimation of a specific RNA share in the total cellular RNA, we propose to chose as internal control mRNA, whose share doesn't change during the total RNA degradation in apoptosis and thus, mRNA degradation is not selective (in relation to other type RNAs). In that way, the real-time RT-PCR method, which is currently the most sensitive and precise method for quantification of RNA in the cell, holds much promise for the investigation of not only different mRNAs, but also small stable RNAs, synthesized by RNA polymerase III.