Charlotte B. Nilsson

CELLULAR RETINOL-BINDING PROTEIN I IS ESSENTIAL FOR VITAMIN A HOMEOSTASIS

The gene encoding cellular retinol (ROL, vitA)‐binding protein type I (CRBPI) has been inactivated. Mutant mice fed a vitA‐enriched diet are healthy and fertile. They do not present any of the congenital abnormalities related to retinoic acid (RA) deficiency, indicating that CRBPI is not indispensable for RA synthesis. However, CRBPI deficiency results in an ∼50% reduction of retinyl ester (RE) accumulation in hepatic stellate cells. This reduction is due to a decreased synthesis and a 6‐fold faster turnover, which are not related to changes in the levels of RE metabolizing enzymes, but probably reflect an impaired delivery of ROL to lecithin:retinol acyltransferase. CRBPI‐null mice fed a vitA‐deficient diet for 5 months fully exhaust their RE stores. Thus, CRBPI is indispensable for efficient RE synthesis and storage, and its absence results in a waste of ROL that is asymptomatic in vitA‐sufficient animals, but leads to a severe syndrome of vitA deficiency in animals fed a vitA‐deficient diet.

Proprioception in Classical Ballet Dancers A Prospective Study of the Influence of an Ankle Sprain on Proprioception in the Ankle Joint

We studied prospectively the influence of ankle sprains on proprioception as measured by recording the pos tural sway of classical ballet dancers. Excellent bal ance and coordination are important for classical ballet dancers, and postural stability requires adequate pro prioception from the ankle joint. Fifty-three professional dancers from the Royal Swedish Ballet, Stockholm, and 23 nonathletes, the control group, participated in the investigation. Postural sway was recorded and an alyzed with a stabilimeter using a specially designed, portable, computer-assisted force plate. Six dancers sustained ankle sprains during followup. The record ings were obtained of these dancers before and after the injuries. The stabilometry results differed among the male and female dancers and the control group as follows: 1) the male dancers demonstrated a smaller total area of sway, and 2) both the male and female dancers had a smaller mean sway on the left foot than on the right (no mean difference in sway was found between the left and right foot in the control group). In comparison with the condition before injury and with the uninjured foot, the postural stability of the dancer was impaired for several weeks after the ankle sprain. Postural stability gradually improved during rehabilita tion and improvement still occured several weeks after professional dancing had resumed.

The Retinoid Signaling System — A Target in Dioxin Toxicity

This review summarizes the available data on the effects of dioxins on retinoid levels, retinoid-related enzyme activities, and toxicological endpoints that have been correlated to retinoid effects. Similarities between dioxin toxicity and retinoid deficiency as well as retinoid excess are pointed out. Several possible levels of interaction between the dioxin and the retinoid signaling pathways are discussed, including the involvement of the Ah receptor, altered retinoic acid homeostasis, and an altered set point for retinoid storage. A hypothesis for the effect of dioxins on retinoids is suggested. In this hypothesis, comprising two cascades of effects on the molecular level, the effect of dioxins on retinoic acid levels is central.

2,3,7,8-Tetrachlorodibenzo-p-dioxin affects retinol esterification in rat hepatic stellate cells and kidney.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic environmental contaminant, interferes with retinoid homeostasis. To elucidate the underlying mechanism, the activities of lecithin : retinol and acyl-CoA : retinol acyltransferase (LRAT and ARAT) were determined in liver, kidney, and hepatic parenchymal and nonparenchymal cell fractions from rats 7 days after a single oral dose of 10 μg TCDD/kg body weight (b.w). Severely depressed LRAT activity in hepatic stellate cells, and greatly increased LRAT activity in kidneys, as well as decreased ARAT activity in stellate cells, were seen in TCDD-treated rats. Although the relevance of decreased ARAT activity under physiological conditions is not clear, the changed LRAT activities most likely contributes significantly to the TCDD-induced effects on tissue retinyl ester levels. It is intriguing that TCDD affects LRAT activity in hepatic stellate cells and kidney in opposite directions. The results suggest that effects of TCDD on retinyl ester tissue levels could be due to a specific interaction with retinoid metabolism.

Use of Model-Based Compartmental Analysis to Study Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Kinetics in Rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic, widespread environmental contaminant that has dramatic adverse effects on the metabolism of vitamin A. We used model-based compartmental analysis to investigate sites and quantitative impacts of TCDD on vitamin A kinetics in rats given on oral loading dose of TCDD in oil (3.5 micrograms/kg) followed by weekly maintenance doses (0.7 microgram/kg) or oil only. [3H]Retinol in its plasma transport complex (experiment 1) of lymph containing chylomicrons labeled mainly with [3H]retinyl esters (experiment 2) were administered i.v., and tracer kinetics in plasma, liver, carcass, urine, and feces were measured for up to 42 days. TCDD treatment caused significant reductions in liver vitamin A levels and significant changes in tracer kinetics and tracer excretion. A four-compartment model was used to fit tracer data for experiment 1; for experiment 2, compartments were added to describe the metabolism of newly absorbed vitamin A. The compartmental models predict that TCDD caused a slight delay in plasma clearance (via an increased recycling to plasma), and in liver processing, of chylomicron-derived vitamin A. Models for both experiments predict that TCDD exposure did not affect the fractional uptake of plasma retinol from the rapidly turning-over extravascular pool, but it doubled the fractional transfer of recycled retinol from slowly turning-over pools of vitamin A to plasma. The residence time for vitamin A was reduced by 70% in TCDD-treated rats, transfer into urine and feces was tripled, and vitamin A utilization rates were significantly increased. Since our results do not indicate that retinol esterification is inhibited, we hypothesize that some of the significant effects of TCDD on vitamin A metabolism result from increased catabolism and mobilization of vitamin A from slowly turning-over pools (especially the liver).

Alzheimer's Disease: Presenilin 2-Sparing γ-Secretase Inhibition Is a Tolerable Aβ Peptide-Lowering Strategy

γ-Secretase inhibition represents a major therapeutic strategy for lowering amyloid β (Aβ) peptide production in Alzheimers disease (AD). Progress toward clinical use of γ-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The γ-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between Aβ production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1 (PS1) over PS2 subclass of γ-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain Aβ levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious γ-secretase targeting strategy for AD.

Spinal sagittal mobility and joint laxity in young ballet dancers. A comparative study between first-year students at the Swedish Ballet School and a control group.

The present study compares spinal configuration, spinal range of motion and joint mobility in first-year students of the Swedish Ballet School and in nondancing students of corresponding age and sex in a state school. The study comprises all the first-year (fourth grade) students (n=23) at the Swedish Ballet School: 11 boys and 12 girls. Their dance practice time was 10 h per week. Thirty-six children in the fourth grade at a state school comprised the control group. None of the controls took ballet classes or participated in organised gymnastics out of school. The neutral spine configuration in standing and the sagittal spine mobility were measured using Debrunners kyphometer and Myrins inclinometer. Joint laxity was measured by employing a modified form of the Contompasis method. Compared with the controls, the dancers showed a higher incidence of joint hypermobility, greater mobility of the thoracic spine, a less prominent lordosis of the lumbar spine and a less prominent kyphosis in the thoracic spine in the neutral standing position. The dancers had done little or no ballet training before entering the ballet school at the age of ten. The results agree with those of earlier studies and suggest that increased flexibility is an asset for those being selected as future ballet dancers.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces lecithin: retinol acyltransferase transcription in the rat kidney

Vitamin A (retinoids) has an essential role in development and throughout life of humans and animals. Consequently, effects of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid metabolism may be contributory to its toxicity. This study was performed to clarify the mechanism behind dioxin-induced retinyl ester formation in the rat kidney. In addition we investigated the possible role of CYP1A1 in dioxin-induced all-trans-retinoic acid (atRA) formation. Male Sprague-Dawley rats were exposed to a single oral dose of TCDD in a combined dose-response and time-course study, with doses ranging from 0.1 to 100 microg/kg bw and time points from 1 to 28 days. Levels of atRA and the expression of two potentially retinoic acid (RA)-controlled proteins critically involved in retinoid storage regulation, lecithin: retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP I), were analyzed in liver and kidney. The expression and activity of cytochrome P4501A1 (assayed as ethoxyresorufin-O-deethylase activity) was assessed to gain insight into its potential role in RA synthesis. There was a significant increase in LRAT mRNA expression in the kidney, whereas no such increase could be observed in the liver, despite significantly increased atRA levels in both tissues. This suggests a tissue-specific regulation of LRAT by TCDD that may be dependent on other factors than atRA. Neither CRBP I mRNA nor protein levels were altered by TCDD. The time-course relationship between CYP1A1 activity and atRA levels in liver and kidney does not exclude a role of CYP1A1 in TCDD-induced RA synthesis. The observed altered regulation of the retinoid-metabolizing enzyme LRAT, together with the low doses and short time required by TCDD to change tissue RA levels, suggest that enzymes involved in retinoid metabolism are specific and/or direct targets of TCDD.

Identification of end points relevant to detection of potentially adverse drug reactions

Expectations are high that the use of proteomics, gene arrays and metabonomics will improve risk assessment and enable prediction of toxicity early in drug development. These molecular profiling techniques may be used to classify compounds and to identify predictive markers that can be used to screen large numbers of chemicals. One of the challenges for the scientific community is to discriminate between changes in gene/protein expression and metabolic profiles reflecting physiological/adaptive responses, and changes related to pathology and toxicology. In these proceedings we provide a brief overview of the technologies with focus on proteomics and the possible applications to mechanistic and predictive toxicology. The discussion also includes strengths and limitations of molecular profiling technologies.

2,3,7,8-Tetrachlorodibenzo-p

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is known to influence vitamin A homeostasis. In order to investigate the mechanism behind this retinoid disruption, male Sprague-Dawley rats were exposed to TCDD at doses ranging from 0.1 to 100 micro g/kg body weight, and were killed 3 days after exposure. Additional groups of rats were killed 1 and 28 days after a single oral dose of 10 micro g TCDD/kg body weight. Serum, kidney, and liver were investigated for retinoid levels, as well as gene expression and enzyme activities relevant for retinoid metabolism. Besides the well known effects of TCDD on apolar retinoids, i.e. decreased hepatic and increased renal retinyl ester (RE) levels, we have found dose-dependent elevation of all- trans-retinoic acid (all- trans-RA) levels in all investigated tissues. In the liver, 9- cis-4-oxo-13,14-dihydro-RA was drastically decreased by TCDD in a dose-dependent manner. In serum, cis-isomers of all- trans-RA, including 9,13-di- cis-RA, were significantly reduced already at the lowest dose level. Protein and mRNA levels of cellular retinol binding protein I (CRBP-I) in liver or kidneys were not significantly altered by TCDD exposure at doses at which retinoid levels were affected, making CRBP-I an unlikely candidate to account for the alterations in retinoid metabolism caused by TCDD. The expression and activities of relevant cytochrome P450 (CYP) enzymes with potential roles in all- trans-RA synthesis and/or degradation (CYP1A1, 1A2, and 2B1/2) were also monitored. A possible role of CYP1A1 in TCDD-induced all- trans-RA synthesis is suggested from the time-course relationship between CYP1A1 activity and all- trans-RA levels in liver and kidney. The significant alteration of the all- trans-RA metabolism has the potential to contribute significantly to the toxicity of TCDD.