Variations in zearalenone activation in avian food species.

Zearalenone (ZEA), a widely distributed oestrogenic fusariotoxin, constitutes a potential risk for human and animal health. ZEA is metabolised to the main metabolites identified in vitro and in vivo: alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL). The efficiency to produce alpha-reduced metabolites appears of particular interest in risk assessment as alpha-reduced metabolites constitute activated forms whereas beta-reduced metabolites are less oestrogenic than ZEA. In this study ZEA activation was compared in avian food species. ZEA and its reduced metabolites were quantified in subcellular fractions of six avian species and rat livers. The alpha-ZOL/beta-ZOL ratio in rats was 19. The various avian food species cannot be considered to be equivalent in terms of ZEA reduction (P<0.001). Quails represented high "beta reducers", with alpha-ZOL/beta-ZOL ratio less than two. Weak "beta reducers" included on one part ducks and chickens showing alpha-ZOL/beta-ZOL ratio greater than 3 and up to 5.6 and on a second part geese, showing a lower production of alpha-ZOL than other poultry. Comparisons of enzyme kinetics in ducks and in quails show that these variations can be explained by the action of various isoforms of dehydrogenases. These results are relevant to food safety, in the context of frequently inevitable contamination of animal feed.

Subchronic dietary exposure of rats to cadmium alters the metabolism of metals essential to bone health.

Cadmium (Cd) was recently identified as a risk factor for osteoporosis. Skeletal damage may be the critical effect of low-level long-term exposure to Cd in the general population exposed via food, but the mechanisms behind this are not clearly understood. We investigated the effect of dietary Cd exposure on metals involved in bone turnover. Female rats received a Cd-supplemented diet (0, 10, 50, or 200 CdCl2 mg/kg diet) for 13 weeks. Cd and essential metals stored in the liver were measured by ICP-MS multianalysis. Mineral content of the livers was modified according to Cd level: iron, magnesium and selenium decreased while copper, zinc and manganese increased with increasing Cd levels. Iron was the most strikingly affected metal, falling to one-fifth of control values at high dietary Cd exposure. In this dosage group, selenium decreased to 36% of mean control concentrations while zinc increased to 168%. This mineral imbalance, especially depleted iron stores, can contribute, at least in part, to the Cd-associated risk of osteoporosis. The association between iron metabolism and Cd exposure should be investigated in humans, as Cd and low iron stores could act synergistically as risk factors for osteoporosis.

Implication of distinct proteins in cadmium uptake and transport by intestinal cells HT-29.

The mechanisms of intestinal absorption have not been clearly elucidated for cadmium, a toxic metal. In this work, we show the implication of distinct proteins in cadmium transport, and the transport step where these proteins are involved. We first validated the HT-29 model by evaluating nontoxic doses of cadmium (ranging from 1 to 20 micromol/L), and by quantifying metal uptake and transepithelial transport. The time-course of 1 micromol/L cadmium uptake at pH 7.5 showed three steps: a rapid one during the first 4 min, probably due to cadmium binding to the membrane; a slower one, characterized by Km of 1.65+/-0.54 micromol/L and Vmax of 3.9+/-0.3 micromol/min per mg protein; and a third, corresponding to slow accumulation that was not equilibrated even after 48 h of cadmium exposure. Intracellular metallothionein content following 1 or 5 micromol/L cadmium exposure showed a significant increase after 6 h of exposure, and was not equilibrated even after 72 h, allowing cadmium accumulation. After 24 h of exposure, metallothionein content was 5-fold, 14-fold, 26-fold, and 50-fold, respectively, for cells grown in the presence of 1, 5, 10, and 20 micromol/L cadmium, compared to control cells. The second step of uptake, characterized by carrier-mediated transport, was markedly increased at pH 5.5, compared to pH 7.5, and strongly inhibited by the metabolic inhibitor dinitrophenol. Moreover Nramp2 transporter cDNA was present in HT-29 cells. These data suggest the involvement of a proton-coupled transporter, which may be the divalent cation transporter Nramp2 (natural resistance-associated macrophage protein 2). Cadmium uptake was also inhibited by copper, zinc, and para-chloromercuribenzenesulfonate (pCMBS), but not by verapamil or ouabain. Taken together, our results indicate that cadmium could enter HT-29 cell by Nramp2 proton-coupled active transport and by diffusion, and accumulates in the cell as long as it binds to metallothionein. Cadmium toxicity could depend partly on the activity of Nramp2, and partly on metallothionein content.

Acetyl- and pseudo-cholinesterase activities of plasma, erythrocytes, and whole blood in male beagle dogs using Ellman's assay.

Organophosphate and carbamate ester insecticides, main causes of pesticide poisoning, inhibit cholinesterase (ChE) enzymes. The aim of this study was to measure and compare baseline values for pseudocholinesterase and acetylcholinesterase (AChE) enzyme activities of different blood fractions in the dog to aid in diagnosis of anticholinesterase poisoning. After collecting blood samples from 23 6-24-mo-old male beagle dogs, Ellman's colorimetric assay was run on plasma, red blood cells (RBC), and whole blood fractions prepared in triplicate. The procedure described in a commercially available kit was applied to plasma and RBC. Hemolyzed whole blood fractions (final dilution 1:8) avoided the time-consuming and laborious separation of plasma and RBC. In addition to the kit substrate acetylthiocholine (ASCh), we used butyrylthiocholine (BSCh) as substrate. Whatever the substrate, ChE activity was lower in RBC than in other blood preparations. It was higher when using ASCh rather than BSCh as substrate (mean IU/L+/-SD): 563+/-144 and 303+/-45 respectively, in contrast to plasma (1640+/-310 and 2510+/-450). Whole blood enzyme activity did not differ significantly according to substrate: ASCh, 1590+/-190; BSCh, 1620+/-250) with a 2 to 3% within-day coefficient of variation. Enzyme activity was significantly lower in dogs <1-y old. This study confirms the low ChE activity in dog RBC compared to other species and other blood fractions. It shows that using whole blood instead of separating RBC from plasma minimizes the variability of ChE activity in the hemoglobin-rich fraction.

Absence of ventral cell populations in the developing brain in a rat model of the Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive condition involving craniofacial and central nervous system malformations with occasional holoprosencephaly (HPE). It is caused by a defect in the 7-dehydrocholesterol (7-DHC) reductase, the enzyme catalyzing the last step of cholesterol biosynthesis. Treatment of pregnant rats with inhibitors of 7-DHC reductase, either AY9944 or BM15.766, has provided a valuable model to study the pathogenesis in SLOS. Recently, cholesterol has been shown to be involved in the post-translational activation of the signaling protein Sonic Hedgehog. To identify the early defects associated with HPE in a rat model of SLOS, and to compare the phenotype of the treated embryos with that of the Shh(-/-) mutants, we examined brain morphology and expression of three developmental genes (Shh, Otx2, and Pax6 ) in 23-somite stage embryos from AY9944-treated dams. We report clearly abnormal morphology of the developing brain, concerning primarily the ventral aspect of the neural tube. We observed a reduced or absent expression of Shh and Otx2 in their ventral domain associated with extended ventral expression of Pax6. The results suggest an absence of the midline ventral cell type at all levels of the cranial neural tube. They provide further evidence that cholesterol-deficiency-induced HPE originates from impaired Shh signaling activity in the ventral neural tube.

Cadmium uptake and transepithelial transport in control and long-term exposed Caco-2 cells: the role of metallothionein.

Exposure of humans to cadmium, a common environmental pollutant, is mainly through food intake. However, the mechanisms of intestinal absorption have not been clearly elucidated for this toxic metal ion. In order to investigate the effects of long-term exposure to this metal and the role of metallothioneins in cadmium absorption, we used human-derived Caco-2 cells cultured on porous membrane filters. We first validated this model by quantifying metal uptake and transepithelial transport on control cells and cells adapted to grow for 2 to 5 weeks in the presence of low doses of cadmium in the culture medium. The nontoxic doses of cadmium (0.1, 1.0, and 5 microM), in which Caco-2 cells could be cultured for many passages without deleterious effects, were determined by evaluating transepithelial resistance of the cells and lactate dehydrogenase leakage. After 24 h of 1 microM Cd exposure, intracellular cadmium levels were 3- and 6-fold higher for cells exposed for extended periods to 1 and 5 microM cadmium, respectively, compared to control cells. In control and long-term exposed cells, this accumulation was inhibited by zinc, copper, and pCMBS, but not by verapamil or ouabain. Intracellular metallothionein content was increased 1.5-, 5-, and 12-fold for the cells grown in the presence of 0.1, 1.0, and 5 microM cadmium, respectively, in the culture medium. The amount of metallothionein synthesized and released by the cells was highly correlated with cadmium accumulation and transport. Our results suggest that Caco-2 cell monolayers are a good predictive model for the study of cadmium intestinal absorption following exposure to repeated low doses of cadmium, and confirm the essential role of metallothionein in the regulation of cadmium intestinal absorption.

Comparative study of cadmium transfer in ewe and cow milks during rennet and lactic curds preparation.

Cadmium transfer from whole milk to cream, rennet, or lactic curds was studied before and following a repeated oral cadmium administration to three lactating ewes and one cow. Before cadmium administration, the cadmium levels in milk were around 0.4 microg/L in ewes and less than 0.2 microg/L in cow. Throughout cadmium administration the mean cadmium levels in milk were 3.3+/-1.4 microg/L in ewes and 2.5+/-1 microg/L in cow. During cadmium administration, 86% of cadmium in ewe milk was dispersed in the skimmed milk and 17% in the cream, whereas only 72% was dispersed in the cow skimmed milk and 27% in the cow cream. Most of milk cadmium was associated with casein fractions. About 70% of milk cadmium was transferred to the rennet or lactic curds of ewe. The remaining cadmium present in whole milk, about 9%, was transferred to the rennet or lactic curd whey. In cow, the proportion of cadmium associated with rennet or lactic curds, rennet curd whey, and lactic curd whey was, respectively, 60%, 56%, 14% and 12% of total milk cadmium. The fraction of total cadmium transferred from milk to its milk products, whatever the species, ranged from 94% to 103%. The factor of concentration of cadmium from whole milk to milk products ranged from three to six. We suggest that the excretion of cadmium into milk is mainly achieved via the milk casein secretion. This is, to our knowledge, the first in vivo study where the cadmium transfer from milk to its milk products after repeated cadmium oral administration to ewe and cow has been studied.

Abnormal cholesterol biosynthesis as in Smith-Lemli-Opitz syndrome disrupts normal skeletal development in the rat.

Smith-Lemli-Opitz syndrome (SLOS) in human infants is a common autosomal recessive malformation syndrome (estimated incidence, 1:20,000). It is characterized clinically by congenital anomalies, especially craniofacial and limb defects, and biochemically by a defect in 7-dehydrocholesterol-delta7-reductase activity (7DHC-reductase), the final enzyme in cholesterol biosynthesis. In previous studies, early administration of the 7DHC-reductase inhibitor AY9944 to pregnant rats resulted in a high frequency of holoprosencephaly, relevant to craniofacial anomalies of SLOS. In order to test the effect of AY9944 on limb development, we treated dams on gestation day 7 (GD7), which delays the biochemical defect to about GD13 to GD14. Sera were sampled on GD12, GD14, and GD21 and cholesterol and dehydrocholesterols (7DHC and 8DHC) were measured by gas-chromatography-mass spectrometry (GC-MS), as for the diagnosis of SLOS. GD21 fetuses were examined for gross malformations and skeletal development. In treated dams, the SLOS biochemical marker 7DHC accounted for one fourth and one third of total sterols, respectively, on GD12 and GD14, and cholesterolemia on these two gestation days was reduced by 50% and 43%, respectively, as compared with control values. This maternal metabolic defect was associated with decrease in fetal weight and delayed ossification. In addition, scapular malformations were observed in four fetuses from three litters. The malformations could have been caused by the same mechanism as holoprosencephaly after early treatment with AY9944. These cholesterol-deficiency-based malformations could have a common cause in the abnormal expression of Hedgehog or other developmental gene proteins, and may thus explain various congenital polymalformative syndromes in humans, including SLOS.

Cholesterol biosynthesis inhibited by BM15.766 induces holoprosencephaly in the rat.

To confirm that blocking 7-dehydrocholesterol delta 7 reductase (7DHC reductase), as observed in Smith-Lemli-Opitz syndrome (SLOS), induces craniofacial defects, we tested BM15.766, which blocks 7DHC reductase but is chemically unrelated to the holoprosencephaly-inducing teratogen AY9944. Rats were given BM15.766 either in methylcellulose from days (D) 1 through D11 (3 treated groups: protocol A) or in olive oil from D4 through D7 (300 mg/kg/d: protocol B). The sera were sampled on D0, D3, and D5 or D6, D10, D14, and D21 to measure cholesterol and dehydrocholesterols in all groups and steroid hormones in protocol B. D21 fetuses showed the holoprosencephaly spectrum of malformations and the treated dams low cholesterol and accumulation of 7DHC, 8DHC, and trienols, as in SLOS-affected children. In the 3 dosage groups the malformations were dose-related and enzymatic cholesterol decreased to a plateau. The DHC reached 25-44% of the total sterols in the dams. In protocol B, one-third of the BM15.766-treated fetuses presented facial malformations and almost two-thirds pituitary agenesis. On D10, cholesterol reached a minimum and the DHC a maximum while estradiol 17 beta and progesterone were lowered, the latter decreasing in correlation with cholesterolemia. A sterol profile similar to that previously observed after AY9944 associated with a similarly high incidence of pituitary agenesis confirmed that time-limited inhibition of 7DHC reductase induces holoprosencephaly and that pituitary agenesis is the minor form of holoprosencephaly.

Inhibition of 7-dehydrocholesterol reductase by the teratogen AY9944: a rat model for Smith-Lemli-Opitz syndrome.

Our aim is to verify the validity of a rat model proposed for Smith-Lemli-Opitz (SLO) syndrome, a developmental disorder characterized by a defect in 7-dehydrocholesterol-delta 7 (7DHC)-reductase and by facial dysmorphism close to the holoprosencephaly caused by the teratogen AY9944. We investigated the sterol profile in rats treated with AY9944 blocking 7DHC-reductase. AY9944 was given orally to rats on gestation day 3 (D3). The sera were sampled for kinetic data on D3, D6, D9, D12, and D21. Cholesterol was measured in parallel by the routine enzymatic method and by the gas chromatography/mass spectrometry (GC-MS) procedure used in SLO diagnosis. In addition to sterols, we dosed steroid hormones punctually on D4 and on D10, and examined D21 fetuses in other animals. The enzymatic method was not specific for cholesterol, and measured 70% pure 7DHC added to a normal serum. On D21, 77% live fetuses showed pituitary agenesis. Cholesterol was rapidly reduced by more than 50% on D6 involving an accumulation of 7DHC, 8DHC, and trienols, as identified in SLO-affected children. The most abundant 7DHC reached a maximum from D9 to D12, equaling cholesterol on D9 (11 mg/dl). On D10, the magnitudes of hypocholesterolemic and of 7DHC accumulation were found to be dose-dependent. Progesterone was reduced as early as 24 hr after treatment and dropped to 40% of the levels in the controls on D10, correlating to the decrease in cholesterolemia. This rat model reproduces the same biochemical perturbations as seen in SLO, strongly suggesting that aberrant sterols (7DHC, 8DHC, or nortrienol) may contribute to the developmental defects.

Changes in serum sterols of rats treated with 7-dehydrocholesterol-delta 7-reductase inhibitors: comparison to levels in humans with Smith-Lemli-Opitz syndrome.

The impaired conversion of 7-dehydrocholesterol to cholesterol, as a result of a permanent inhibition of the activity of 7-dehydrocholesterol-delta 7-reductase, has been reported in the Smith-Lemli-Opitz (SLO) syndrome (1, 2). For the purpose of experimental teratology, an animal disease model consisting of the offspring of pregnant rats treated with AY 9944 or BM 15766, inhibitors of 7-dehydrocholesterol-delta 7-reductase, was established. The present study compares the profiles of sterols in rat serum, obtained after transient treatment with inhibitors, with profiles of sterols obtained from patients with the permanent enzyme defect. AY 9944 (single dose of 50, 75, or 100 mg/kg) or BM 15766 (60, 75, or 90 mg/kg per day for 11 days) induces hypocholesterolemia and accumulation of 7-dehydrocholesterol and aberrant sterols in rat serum. The aberrant sterols in the treated rats are similar to those detected in human SLO patients by gas chromatography coupled to mass spectrometry (1, 3, 4) and were identified as 7- and 8-dehydrocholesterol, two trienols (I and II), and 19-nor-5,7,9(10)-cholestatrien-3 beta-ol. The time- and dose-dependences of the biochemical alterations are compared to the teratogenic abnormalities induced by inhibitors. The dietary cholesterol supplementation that suppresses embryo malformations induced by AY 9944 prevents severe hypocholesterolemia and decreases the aberrant sterol levels. As a function of time after intoxication, the 8-dehydrocholesterol to 7-dehydrocholesterol ratio increases, suggested that 8-dehydrocholesterol is derived from the gradual conversion of the accumulated 7-dehydrocholesterol. The ratio of 8-dehydrocholesterol to 7-dehydrocholesterol is higher in human SLO than in the animal disease model. This may be explained by a permanent block in 7-dehydrocholesterol-delta 7-reductase in SLO compared to a transient inhibition of this enzyme in the animal model.

Effects of fluoride on secretory and postsecretory phases of enamel formation in sheep molars.

Effect of fluoride was assessed on molars during and after mineralization. Two groups of 7 sheep each were dosed orally with 3.5 mg of fluoride/kg of body weight daily for 4 months (from 5 to 9 months after birth). Sheep of the first group were slaughtered immediately after fluoride administration; those of the second group were slaughtered 4 months later at the age of 13 months. Three control groups of 7 sheep each were slaughtered at 5 months (to determine the state of the teeth at the beginning of fluoride administration), and at 9 and 13 months. During fluoride administration, plasma fluoride concentration rapidly increased to about 0.50 microgram/ml; after fluoride administration, it stabilized at 0.20 microgram/ml in treated sheep, whereas controls had concentration of 0.10 micrograms/ml (P less than 0.01). Parts of the molars that were in the process of mineralization during fluoride administration (mainly second molars) had thinning enamel, with pits, mainly close to the apex, marked decrease in hardness throughout the layer (less than 100 Vickers U, compared with 240 Vickers U), and fluoride accumulation twice as high as that in controls (1,000 to 2,500 mg/kg [dry weight]). Fluoride accumulation was higher in dentine (2,700 to 4,200 mg/kg), but hardness was less affected. On parts of the molars that were already mineralized (mostly, the first molar), changes in the appearance of enamel and cementum, decreased hardness (less important than in teeth during mineralization) affecting outer enamel more than inner enamel, high fluoride concentration (4,000 to 5,500 mg/kg [dry weight]) in outer enamel extending over 200 microns were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoride pharmacokinetics in the ewe: a linear pharmacokinetics model.

The kinetics of fluoride was studied in a group of 3 adult ewes given sodium fluoride solutions at 3 dose levels (0.15, 0.375 and 0.75 mg/kg bw). Data were analysed using both compartmental and noncompartmental approaches. A 3-compartment open model was selected to describe the data. Comparison of the different parameters indicated an absence of change with dose level, suggesting that fluoride behaved linearly at the doses under study. The half-life of elimination was 2.57 +/- 1.28 h, the steady-state volume of distribution was 0.26 +/- 0.5 L/kg and the body clearance was 0.105 +/- 0.26 L/kg/h. It was concluded that a single intravascular fluoride administration (bolus) may be used to evaluate oral bioavailability of fluoride in sheep in toxicity studies.

[Soil fluoride: low digestive absorption in sheep]. / Fluor du sol: faible absorption digestive chez les ovins.

The percentages of digestive absorption of fluoride contained in various soils were assessed by the balance technique in groups of 4 ewes given soil enriched concentrated feed. Four soils collected in an area close to a source of fluoride and 3 samples from non polluted sites were used. The levels of extractable fluoride were significantly higher in the polluted soils. The various soils have little influence on the amounts of fluoride excreted in urine; these amounts are not correlated with the amounts of fluoride absorbed in the digestive tract. The percentages of digestive absorption of soil fluoride ranged between 4.5 and 23%, with 4 values close to 20%. The values are correlated with the soil total fluoride concentration and not with the soil extractable fluoride concentration.

Indicators of lead, zinc and cadmium exposure in cattle: II. Controlled feeding and recovery.

Two pre-exposed and 2 normal heifers were fed lead (Pb), zinc (Zn), and cadmium (Cd) polluted hay (500 g/100 kg body weight) over a 17-week period. They were then examined over a 10-month period (42 or 38 weeks) to study the decay of the indicators of exposure. The elimination pattern of blood Pb and Zn protoporphyrin concentrations displayed a very slow decay. A bi-exponential equation, with the half-times of the fast component set at approximately 1 week, and the half-times of the slow component set from 3 mo to 2 years, was fitted to blood Pb levels. In man, the half-life of the slow component is still longer (2 to 15 years). This slow elimination rate represents release of Pb from skeleton, which in bovines, may accumulate up to 100 ppm Pb/dry or more. Lead concentrations in the hair were not proportionate to the areas under the curves of blood Pb levels; there was the same lack of correlation concerning skeleton and viscerae Pb levels. The withdrawal of contaminated hay from the diet resulted in a significant increase in blood copper. This is in accordance with the depressive effect of Pb and Zn on the bioavailability of this metal.