Assembling the prenylneoflavone system through a Pechmann condensation/Mitsunobu reaction/Claisen rearrangement/olefin cross-metathesis sequence.

Abstract: The multistep synthesis of a prenylneoflavone through a sequence of the Mitsunobu reaction/Claisen rearrangement/olefin cross-metathesis reaction has been accomplished in 5% yield over six steps starting from commercially available 3-methoxyacetophenone. The sequence is shown to be compatible with a Pechmann condensation which proved to be a robust and cost-effective method for the assembling of the α-pyrone core. The results open doors to a general approach to the prenylneoflavone system starting from phenol and acetophenone derivatives.

Modulation of soy isoflavones bioavailability and subsequent effects on bone health in ovariectomized rats: the case for equol.

INTRODUCTION: Soy products are of particular interest because of their potential health benefits in a range of hormonal conditions, such as osteoporosis, due to their high content in phytoestrogens. Because equol, the main metabolite from soy isoflavones, is thought to be powerful, the present study was designated to evaluate the bone-sparing effects of equol by either providing the molecule through the diet or by eliciting its endogenous production by modulating intestinal microflora by short-chain fructooligosaccharides (sc-FOS) or live microbial (Lactobacillus casei) together with daidzein, its precursor. METHODS: A comparison with daidzein and genistein was also performed. Rats (3 months old) were ovariectomised (OVX) or sham-operated (SH). Ovariectomised rats were randomly assigned to six experimental diets for 3 months: a control diet (OVX), the control diet supplemented with either genistein (G), or daidzein (D), or equol (E) at the level of 10 microg/g body weight/d. The remaining OVX rats were given daidzein at the dose of 10 mug/g body weight/d, simultaneously with short-chain FOS (Actilight, Beghin-Meiji) (D+FOS) or Lactobacillus casei (Actimel, Danone) (D+L). The SH rats were given the same control diet as OVX. RESULTS: Genistein, daidzein or equol exhibited a bone sparing effect. Indeed, total femoral bone mineral density (BMD) was significantly enhanced (compared to that of OVX rats), as was the metaphyseal compartment. Bone strength was improved by E consumption, but not by genistein or daidzein given alone. As far as the FOS diet is concerned, the addition of prebiotics significantly raised efficiency of the daidzein protective effect on both femoral BMD and mechanical properties. The effects of lactobacillus were similar, except that the increase in metaphyseal-BMD was not significant. CONCLUSION: In conclusion, long-term equol consumption, like genistein and daidzein, in the ovariectomized rat, provides bone sparing effects. Adding indigestible sugars, such as FOS or live microbial as L. casei, in the diet significantly improves daidzein protective effects on the skeleton.

Disposition of genistein in rainbow trout (Oncorhynchus mykiss) and siberian sturgeon (Acipenser baeri).

Genistein (G) is a xenoestrogen from soy present in fish diet. In vivo, a 50-fold difference in sensitivity to genistein on vitellogenin (VTG) synthesis was found when comparing trout and sturgeon. This difference was not linked to the estrogen receptor affinity nor to the sensitivity of induction of the VTG pathway. The study was performed to check if differences in the G disposition in the two species could explain their difference of sensitivity to G. A pharmacokinetic analysis of radiolabeled G was performed to determine its bioavailability and metabolism in both species. G was used at levels corresponding to fish farm exposure. G plasma levels after chronic ingestion were found to be 15.6 times higher in sturgeon than in trout. Sturgeon primarily produces sulfate conjugates after G ingestion whereas trout mainly produces glucuronides. Sturgeon was able to excrete orobol glucuronide in bile. An important first pass effect was suggested in both species. No accumulation of G or its metabolites was observed in the two species. Trout muscles accounted only for 0.14 of radioactivity 48 h post-ingestion similarly to sturgeon. Trout viscera accounted for 15% of the radioactivity 48 h post-ingestion. In sturgeon, 48 h post-ingestion, viscera accounted for 21.5% of the radioactivity. These rates decreased rapidly thereafter. The study partly explains the difference in sensitivity to G, previously recorded between the two species. In addition, it shows that human exposure to G through farmed fish consumption is negligible.

Concentrations of isoflavones in plasma and urine of post-menopausal women chronically ingesting high quantities of soy isoflavones.

Soy food or food supplements based on soy containing isoflavones (Isos) are increasingly available in Western countries. However, the variability of Isos levels in urine and plasma in humans during chronic ingestion is poorly documented. Nevertheless, this is the way these compounds will most probably be used in the future, especially if the soy-based supplements market goes on increasing. Here, glycosilated Isos in an enriched extract of Prevastein equal to 100 mg of equivalent Isos aglycone was given daily to 27 post-menopausal women for 30 days and to 12 post-menopausal women for 60 days. Volunteers were given Prevastein in a cereal bar (25 mg Isos) and in a yoghurt (25 mg Isos) both at breakfast and dinner. Plasma samples were collected after overnight fasting. Urine samples were aliquots of a 24 h collection checked on volume and creatinin excretion levels. Genistein, daidzein and equol were measured at day 0 and every 15 days afterwards, using original specific ELISAs. Constant levels were reached from the 15th day. About 59.2% of the volunteers were significant equol producers in the first experiment and 58.3% in the second. A large variability in plasma and urine levels was observed among post-menopausal women consuming 100 mg Isos per day, although remaining relatively stable in each individual subject. This could partly account for the controversial effects of Isos recorded so far in clinical studies. So Isos plasma levels would have to be assayed during chronic exposures, and could help to better understand the large variability of the effects classically observed in clinical studies. ELISA techniques could be easily exported to analytical laboratories to help physicians and nutritionists with their prescriptions.

Fructooligosaccharides maximize bone-sparing effects of soy isoflavone-enriched diet in the ovariectomized rat.

Isoflavones (IF) have been increasingly implicated for use in the prevention of osteoporosis. As their bioavailability could be improved by modulating intestinal microflora, the present study was undertaken to investigate whether IF and fructooligosaccharides (FOS), which are known to modify large-bowel flora and metabolism, may exhibit a cooperative bone-sparing effect. This work was carried out on 3-month-old Wistar rats assigned to 12 groups: 2 SH (sham-operated) and 10 OVX (ovariectomized). Animals received a diet for 90 days containing total IF (Prevastei HC, Central Soya) at 0 (OVX and SH), 10 (IF10), 20 (IF20), 40 (IF40), or 80 (IF80) microg/g body weight per day. FOS (Actilight, Beghin-Meiji) were orally given to half of the groups, (OVX FOS), (IF10 FOS), (IF20 FOS), (IF40 FOS), (IF80 FOS), and (SH FOS). Isoflavones exhibited a bone-sparing effect as soon as consumption reached 20 microg/g/day, whereas only the highest dose induced a weak uterotrophic activity. Indeed, total femoral bone mineral density (BMD) was significantly enhanced (compared with that of OVX rats), as was the metaphyseal compartment. Bone strength was improved as well. As far as the FOS diet is concerned, addition of prebiotics significantly raised the efficiency of the IF protective effect on both femoral BMD and mechanical properties. The trend toward higher BMD levels with the lowest IF dose (IF10) even reached a significant level when FOS were added. This effect could be explained by a reduced bone resorption. In conclusion, daily IF consumption prevented castration-induced osteopenia by decreasing bone resorption when given at 20, 40, or 80 microg (total isoflavones)/g/day. Simultaneous FOS consumption improved IF protective effect on the skeleton, with the lowest IF dose becoming efficient. Enhancement of IF bioavailability, following FOS fermentation, is probably involved.

Screening for oestrogenic activity of plant and food extracts using in vitro trout hepatocyte cultures.

The use of in vitro trout hepatocyte cultures is shown to provide a simple and effective way to screen plant and food products for oestrogenic activity. The relative oestrogenic activities of 0.1 g each of extracts of phytosterol, soy isoflavone, red clover, kudzu and soybean extracts were determined using this assay and found to be equivalent to 212, 1, 3.2, 132 and 1025 nM of 17beta-estradiol, respectively. Controls were performed on soybean and kudzu extracts using specific ELISAs for isoflavones and these confirmed the validity of the cell culture assay. The method described offers an advantage over current methods in that it can detect increased oestrogenic activity that may occur as a result of metabolic activation of pre- or pro-oestrogens liver cells.

Binding affinities of hepatic nuclear estrogen receptors for phytoestrogens in rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baeri).

Phytoestrogens are dietary estrogenic contaminants capable of inducing vitellogenin synthesis in rainbow trout and Siberian sturgeon. A competitive-binding assay on their hepatic estrogen receptors (ER) was performed to determine the relative affinity of phytoestrogens compared to estradiol (E(2)). Phytoestrogen concentrations used were 1000 times higher than for E(2), except for genistein and formononetin. For each compound, the competition with 50%-bound labelled E(2) (DC(50)) was considered in order to classify phytoestrogens according to their affinity for ER. The affinities are compared for each species. In rainbow trout, estradiol (DC(50): 7 nM)>formononetin (DC(50): 260 nM)>genistein (DC(50): 570 nM)>equol (DC(50): 5.3 microM)>daidzein (DC(50): 9 microM)>biochanin A (DC(50): 100 microM). In sturgeon, estradiol (DC(50): 5 nM)>genistein (DC(50): 220)>formononetin (DC(50): 1 microM)>equol>(DC(50): 8.3 microM)>daidzein>(DC(50): 80 microM)>biochanin A (DC(50): 100 microM). These results demonstrate that phytoestrogens, mimicking estradiol, can disturb the endocrine system by competing for ER. Also, the higher sensitivity to genistein observed in vivo in Siberian sturgeon (vitellogenin synthesis), compared to rainbow trout, is not due to a higher affinity of genistein for the hepatic ER. Thus, the metabolism of phytoestrogen could be species dependent and affect sensitivity.

Effects of dietary phytoestrogens in vivo and in vitro in rainbow trout and Siberian sturgeon: interests and limits of the in vitro studies of interspecies differences.

A study of the effects of dietary genistein on trout and sturgeon in vivo showed that sturgeon was sensitive to 20 ppm of genistein, whereas trout was not. To analyze the origin of this interspecies difference in sensitivity, a cell culture technique was developed with hepatocytes from sturgeon and compared to results obtained with hepatocytes from trout in the same system. The hepatocyte culture proved to be useful as bioassay for estrogenicity. Vitellogenin (VTG), assayed by a specific enzyme-linked immunosorbent assay, was used as a biomarker of the estrogenic activity. 17 beta-Estradiol, its glucuronide and sulfate derivatives, and estradiol analogues (ethynylestradiol and diethylstilbestrol) were tested. Nonestrogenic compounds such as androgens, progesterone, and cortisol were tested as negative controls. VTG production was monitored at doses ranging from 1 nM to 10 microM estradiol. Phytoestrogens, from the isoflavone family, were tested individually at increasing doses exhibiting dose response curves for concentrations from 500 nM to 10 microM. With tamoxifen, an antagonist of estrogen receptors, the estrogenic effect was partially reduced. The effect was the same with ICI182,780 in sturgeon, whereas the effect was the opposite in trout. The estrogenic potency of the isoflavones ranged differently between the two species in the following order: biochanin A < daidzein = formononetin < genistein < equol in trout and biochanin A < genistein < daidzein < formononetin < equol in sturgeon. Further, in sturgeon, formononetin was the most potent phytoestrogen in vitro, whereas its activity was weakest in vivo. These data suggest that one must reconsider the relevance of heterologous estrogenic tests and of homologous in vitro tests for estrogenic potency of chemicals.

Dose-dependent bone-sparing effects of dietary isoflavones in the ovariectomised rat.

The dose-dependent bone-sparing effects of dietary isoflavones (IF) were investigated in adult (7-month-old) Wistar rats. Forty animals were ovariectomised, allocated into four groups of ten rats each, and immediately treated orally with IF at 0 (OVX), 20 (IF20), 40 (IF40) or 80 (IF80) microg/g body weight per d for 91 d; ten sham-operated (SH) controls received the same diet without added IF. Animals were killed on day 91. Both femoral failure load and total femoral, diaphyseal or metaphyseal bone mineral densities (BMD) were lower in OVX animals than in SH animals. Urinary deoxypyridinoline (DPD) excretion, a marker of bone resorption, and plasma osteocalcin (OC) levels, a marker of osteoblast activity, were higher in OVX animals than in SH animals. Total femoral and diaphyseal BMD and femoral failure load were similar in IF-treated rats and SH rats. Although metaphyseal BMD in IF40 or IF80 rats was similar to that in SH rats, its value was lower in IF20 rats than in controls. The day 91 urinary DPD excretion in IF40 and IF80 rats, but not in IF20 rats, was similar to that in SH rats. Day 91 plasma OC concentrations in IF-treated rats were similar to day 45 values, but were decreased in OVX and SH rats. Thus, daily IF consumption prevented ovariectomy-induced bone loss, both by depressing bone resorption and stimulating osteoblast activity. Moreover, as only the highest IF level induced a weak uterotrophic activity, the optimal IF dose which preserves both cancellous and cortical bone, but exhibits no oestrogen-like effects on the uterus, was 40 microg/g body weight per d.

Soybean isoflavones dose-dependently reduce bone turnover but do not reverse established osteopenia in adult ovariectomized rats.

We assessed the dose-dependent effects of daily soybean isoflavone (IF) consumption in reversing bone loss in adult ovariectomized rats. On d 0, female Wistar rats (7 mo old; n = 55) were either sham-operated (SH; n = 14) or ovariectomized (n = 41). On d 80, intermediate rats (SH: n = 5; ovariectomized: n = 5) were killed to confirm the ovariectomy-induced bone loss. The remaining ovariectomized rats were randomly assigned to one of four groups of nine rats each and fed soybean IF (mixed with a soy protein-free semipurified diet) at 0 (OVX), 20 (IF20), 40 (IF40) or 80 (IF80) mg/(kg body. d) for 84 d. Simultaneously, SH rats were fed the semipurified diet without any additional compound and killed on d 164, as were the other rats. As expected, both bone mineral density in the total femur and in its diaphyseal and metaphyseal subregions and cancellous bone area/measured surface in the distal femur metaphysis were lower in OVX than in SH rats (P: < 0.05). OVX rats had higher plasma osteocalcin concentration and urinary deoxypyridinoline excretion than SH rats (P: < 0.05). On d 164, osteocalcin and deoxypyridinoline concentrations were lower in IF40 or IF80 rats than in OVX rats (P: < 0.05). Nevertheless, neither bone mineral density nor cancellous bone area was greater in IF-fed rats than in OVX rats. Therefore, in adult ovariectomized rats, daily soybean IF consumption decreased bone turnover but did not reverse established osteopenia.

Effect of genistein-enriched diets on the endocrine process of gametogenesis and on reproduction efficiency of the rainbow trout Oncorhynchus mykiss.

Three practical diets were formulated to contain 0, 500, or 1000 ppm genistein. The three diets were distributed for 1 year to groups of rainbow trout undergoing their first gametogenesis and until spawning. Growth performance of rainbow trout was not affected by dietary treatments. Plasma cholesterol levels were equivalent between groups. In males, a slight but constant induction of vitellogenin (VTG) synthesis and a decrease in testosterone levels were observed. A slight decrease in plasma levels of betaFSH and betaLH was noticed at the end of spermatogenesis in the male fish fed a diet with 500 ppm (genistein) (from 2.16 +/- 0.39 to 1.47 +/- 0.23 for betaFSH and from 0.44 +/- 0.09 to 0.31 +/- 0.09 for betaLH). There was a significantly reduced 17alpha,20beta(OH)(2)-progesterone (from 10.93 +/- 0.88 in control to 5.46 +/- 0.92 in males and from 251.22 +/- 21.40 to 183.22 +/- 13.48 in females). Testicular development was accelerated in genistein-fed fish, and sperm motility and concentration were decreased in a dose-dependent manner at spawning. In females, a significant increase in plasma VTG occurred only at the beginning and at the end of oogenesis. Testosterone levels were decreased at the beginning of oogenesis. Both betaFSH and betaLH were decreased by genistein (from 6.38 +/- 1.55 to 3.44 +/- 0.82 for betaFSH and from 15.18 +/- 3.00 to 6.93 +/- 0.99 for betaLH in females), whereas spawning was delayed only in females fed the diet with 500 ppm of genistein. Gamete quality was impaired only in this group, as underlined by a lower percentage of ovulating females (from 100 to 79% at the end of the trial), a lower fertilization rate, and a lower viability of fry. These results may be explained by the agonistic/antagonistic effect of genistein on estrogen function related to the tissue ratio between endogenous estrogens/genistein.

Daidzein is more efficient than genistein in preventing ovariectomy-induced bone loss in rats.

We investigated the ability of genistein and daidzein, two soybean isoflavones, compared with that of 17 alpha-ethinylestradiol, to prevent bone loss in ovariectomized rats, a model for postmenopausal osteoporosis. Female Wistar rats (n = 65; 12 mo old) were either sham-operated (SH; n = 13) or ovariectomized (OVX; n = 52). On d 0, OVX rats were randomly assigned to groups as follows: 13 received genistein [G; 10 mcg/(g body weight. d)], 13 were treated with daidzein [D; 10 mcg/(g body weight. d)], 13 received 17 alpha-ethinylestradiol [E(2); 30 mcg/kg body weight. d)] and 13 were untreated (OVX). Compounds were mixed with a soy protein-free powdered semipurified diet and given orally for 3 mo. On d 90, the bone mineral density (BMD) in lumbar vertebrae, femur and its metaphyseal and diaphyseal zones (rich in cancellous and cortical bone, respectively) was lower in OVX than in SH (P < 0.01). In D or E(2), the four BMD were not different from SH, whereas in G, only the diaphyseal BMD was not different from SH. Image analysis performed in the distal femur metaphysis revealed that the cancellous bone area was lower in OVX than in SH (P < 0.01). Only the area in D was not different from that in SH. Finally, the bone turnover, which was higher in OVX than in SH (P < 0.005 and P < 0.05 for plasma osteocalcin concentration and urinary deoxypyridinoline excretion, respectively), was not different in G, D or E(2) compared with SH. Therefore, consumption of 17 alpha-ethinylestradiol or daidzein was more efficient than genistein in preventing ovariectomy-induced bone loss in rats.

Synthesis of haptens and conjugates for ELISAs of phytoestrogens. Development of the immunological tests.

Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. The different haptens were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for specific antibodies. IC(50) values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3 and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well as in biological fluids after a separation step. These ELISAs should be valuable also in the food industry to control phytoestrogen concentrations prior to and after processing.

Plasma vitellogenin levels during the annual reproductive cycle of the female rainbow trout (Oncorhynchus mykiss): establishment and validation of an ELISA.

Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric from of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20-320 ng/ml (80-20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.

Effects of flavonoids on aromatase activity, an in vitro study.

In the study, the inhibitory effect of flavonoids, including isoflavonic phytoestrogens, on the ovarian aromatase enzyme complex from the rainbow trout, Oncorhynchus mykiss, was assessed in vitro. Some of the compounds tested on fish were also tested on human placental aromatase activity as a comparison between the two sources of enzyme. It was found that flavone, dl-aminoglutethimide, apigenin, quercetin, 7,4'- dihydroxyflavone, alpha-naphthoflavone and equol were potent inhibitors of the ovarian aromatase activity in rainbow trout. Relative potencies (RP) of these compounds compared to flavone (assigned an effect of 1) were, respectively, 19.0, 8.7, 5.3, 3.7, 3.2 and 0.9. Two other phytoestrogens, namely biochanin A and genistein, slightly inhibited aromatase activity. Finally, 7-hydroxyflavone, formononetin, daidzein, coumestrol, chrysin, flavanone and estradiol-17beta did not inhibit ovarian aromatase activity at doses up to 1000 microM. Experiments on human placental aromatase showed inhibitory effects of dl-aminoglutethimide, flavone, flavanone and equol with RP values of 2.8. 1, 1.5 and 0.4, respectively. These results are in accordance with previous studies. The influence of the experimental procedure on IC50 values and RP is discussed.

Interactions between phytoestrogens and human sex steroid binding protein.

The interactions of human Sex steroid binding protein (SBP) and the lignans [Nordihydrogaiaretic acid (NDGA) enterolactone (Ent), enterodiol (End)] and isoflavonoid phytoestrogens [Equol (Eq), diazein Dad), genistein (Gen)] were studied. The phytoestrogens had different dose-dependent inhibitory effects on steroid binding by SBP. Their relative efficiencies were: Ent> or = NDGA = Eq > Gen for displacing E2 and Eq > Ent > NDGA > Gen for displacing T. End and Dad were much less active. Scatchard analysis suggested that NDGA had similar non- competitive effects on T and E2 binding by reducing the number of binding sites without changing the association constants. But Eq seemed to inhibit E2 binding non-competitively and T binding competitively. NDGA binding to SBP reduced the immunorecognition of SBP by monospecific anti-SBP antibodies, suggesting that NDGA changed SBP immunoreactivity. Unlike NDGA, Eq binding to SBP caused no immunological changes in SBP, indicating qualitative differences in the effects of the lignan and isoflavonoid. Our results indicate that phytoestrogens may modulate the SBP activity and so influence the role of this protein in the delivery of hormonal information to sex steroid-dependent cells.

Occurrence and in vitro biosynthesis of 11-ketotestosterone in Siberian sturgeon, Acipenser baeri Brandt maturing females.

High levels of 11-ketotestosterone (11KT) were found (49 to 160 ng ml(-1)) in plasma of Siberian sturgeon females during the end of their reproductive cycle. These levels were measured either by specific radioimmunoassay, or both by specific radioimmunoassay and by UV absorption after HPLC (isocratic conditions, 33% methanol, 26% acetonitrile, 41% water). In order to find the origin of 11KT synthesis, ovaries were incubated (30 min and 2h at 20°C) with tritiated 17-hydroxyprogesterone (17OHP) or with tritiated androstenedione (A4). Testosterone (conversion rate from tritiated 17OHP: 4%) and 11-ketotestosterone (conversion rate from tritiated A4: 1.6%) were identified as metabolites of respectively 17OHP and A4 (TLC, HPLC and crystallization). 11ß-hydroxyandrostenedione (11ßOHA4) and 11ß-hydroxytestosterone (11ßOHT) were suggested to be intermediate metabolites. Besides interrenal and blood cells were incubated respectively with tritiated cortisol and tritiated A4. 11ßOHA4 was identified in interrenal incubation (yield from tritiated cortisol: 1.2%). 11KT in interrenal (yield from tritiated cortisol: 0.14%), and 11ßOHA4 and 11KT in blood cells (yield from tritiated A4: 1.6%), were suspected to be synthesized (TLC, HPLC, acetylation). No significant metabolization of tritiated cortisol could be found in liver. The possible contribution of each of these tissues to high 11KT levels found in plasma is discussed.

Monolayer and aggregate cultures of rainbow trout hepatocytes: long-term and stable liver-specific expression in aggregates.

An aggregate culture system for rainbow trout hepatocytes was developed to study liver-specific mRNA expression. Maintenance of differentiated functions and morphology of hepatocytes were examined using both monolayer and aggregate systems. The rainbow trout estrogen receptor and vitellogenin genes were induced by estradiol and their mRNAs used as markers of cell differentiation during cell culture. In monolayer culture, rainbow trout hepatocytes established very few cell-cell contacts in minimal media. The use of more complete media promotes cell-cell contacts and cell islet formation. Hepatocyte response to estradiol stimulation was generally lower than in vivo but a correlation between the degree of cellular organization and the intensity of the hormonal response was observed. However, in this system hepatocytes progressively lost their specific hormonal response between 5 and 10 days. In aggregates with DMEM/F12 and Ultroser SF, cell-cell contacts were maximized and stabilized during at least one month. The levels of rainbow trout estrogen receptor and vitellogenin mRNAs induced by estradiol were stable and maintained at a level comparable to in vivo levels; vitellogenin synthesis and secretion remained fully functional for the duration of the culture.

Seroepidemiology, morbidity and vaccination strategies against rubella infection. Eight years experience in Oltrepò Pavese.

Selective rubella vaccination of schoolgirls in Italy started 14 years ago following the United Kingdom strategy that was adopted in 1970. The aims of this program were to eliminate the risk of rubella among women of childbearing age, encourage the acquisition of immunity by natural infection during early childhood and allow the vaccine-induced antibody production by the circulating virus. On the basis of this program, between 1982 to 1990, a prospective serosurvey for rubella antibody in the province of Pavia was performed. The results showed a decline in the overall seropositivity rate for rubella antibodies from 57.7% in 1982 to 41.9% in 1984 followed by a remarkable increase in 1985 (53.3%) and in 1987 (56.5%). This trend was confirmed by the number of cases reported to the local Public Health Service. The results of this study provide further evidence of the need to change the current selective immunization policy in order to obtain a significant reduction of risk of the infection in the population.