Studies on the pro-oxidant chemistry of flavonoids

There is currently much interest in the development of functional foods aiming at the prevention of the development of some diseases, for example cancer, by the introduction of selected natural substances at elevated levels into the diet. The rationale for this approach is based especially on epidemiological data that indicate that food items containing such chemicals may reduce the risk of these diseases in humans. Epidemiological studies indicate, for example, that diets rich in fruit and vegetables protect against a variety of diseases, including heart diseases and certain forms of cancer. However, identification of the actual ingredient in a specific diet responsible for the beneficial health effects remains an important bottleneck for translating observational epidemiology to development of a functional food ingredient. The protection against cancer afforded by fruit and vegetables has been attributed to antioxidant micronutrients such as vitamin C, beta-carotene and vitamin E, which may act at many sites, including the stomach, intestine, lung and bladder. However, present scientific attention is focusing as well on the significance of other minor dietary components, notably the flavonoids as protectants against disease. Flavonoids are widespread in nature and are found in considerable quantities in fruits, vegetables, seeds, peel and tubers. The average Western diet may provide up to 1 g of flavonoids per day. Numerous in vitro studies show that flavonoids are potent antioxidants and metal chelators. Their potential as anti-inflammatory, antiallergic and antiviral compounds has also attracted attention. These studies provide the basis for the present rapidly increasing interest for the use of flavonoids as functional food ingredients. As a result increased human exposure to flavonoids can be expected in the near future. In shops and at the internet, food and food supplements based on (iso)flavonoids as functional ingredients are marketed. This, although hard scientific data supporting the health claims as well as data allowing a balanced risk-benefit evaluation are lacking. For flavonoids increased future human exposure regimens induce the question on their pro-oxidant chemistry. There is considerable evidence that some flavonoids are mutagenic in both bacterial and mammalian experimental systems. A high incidence of gastric cancer in some human populations has been linked to consumption of wine containing potentially mutagenic flavonoids (Tamura et al. , Proc. Natl. Acad. Sci. USA. 77, 4961-4965, 1980, Hoey et al. , Am. J. Epidemiol., 113, 669-974, 1981). Relatively little is understood about either the toxicity or protection afforded by flavonoids in humans.Since flavonoid quinone/quinone methides have been suggested as the major metabolites responsible for the possible pro-oxidant toxicity and mutagenicity of flavonoids, characterisation of flavonoid quinone chemistry is of importance. However, little information is available on the structure and reactivity of these flavonoid oxidation products. Therefore, the objective of this thesis was to investigate the pro-oxidant chemistry of flavonoids and to perform structure activity studies on the chemical behaviour of 3',4'-dihydroxyflavonoids with special emphasis on the nature and reactivity of the quinone/quinone methide type metabolites formed. Using the GSH trapping method, HPLC, LC/MS, MALDI-TOF, 1H NMR, 13C NMR and quantum mechanical computer calculations the quinone/quinone methide chemistry of a series of 3',4'-dihydroxyflavonoids could be characterised.The results provide insight in structure-activity-relationships for the pro-oxidant chemistry of these electrophilic quinone/quinone methide flavonoid metabolites. The results obtained also reveal an unexpected pH-dependent electrophilic behaviour of B ring catechol flavonoids. Furthermore the results of this thesis also reveal, for the first time, evidence for the pro-oxidative chemistry of quercetin in a cellular in vitro model. The formation of these glutathionyl-flavonoid adducts provides evidence for the actual pro-oxidative formation of reactive quinone type metabolites from B ring catechol flavonoids in the selected cellular in vitro model using melanoma cells. Oxidation of the catechols to quinones and their isomeric quinone methides generates potent electrophiles that could alkylate DNA. Interestingly, the structural requirements essential for good antioxidant activity match the requirements essential for pro-oxidant action and quinone methide formation. Altogether, the pro-oxidant behaviour of flavonoids and their quinone/quinone methides are far from straight forward and need to be re-evaluated especially in the framework of the risk-benefit evaluation of the use of these flavonoids as functional food ingredients and/or food supplements.SamenvattingEr is momenteel veel interesse voor de ontwikkeling van functionele voedingsmiddelen (functional foods), met als doel het voorkomen van het ontstaan van ziekten zoals bijvoorbeeld kanker, via het in verhoogde mate introduceren van geselecteerde natuurlijke bestanddelen in het dieet. De basis voor deze aanpak wordt momenteel met name gevonden in epidemiologische studies die laten zien dat diëten rijk aan specifieke voedselcomponenten of ingrediënten de kans op bepaalde ziekten bij de mens verlagen. Zo geven epidemiologische studies bijvoorbeeld aan dat diëten die rijk zijn aan fruit en groenten beschermen tegen een aantal ziekten zoals hartziekten en bepaalde vormen van kanker. Echter, het identificeren van de belangrijke ingrediënten in het betreffende dieet die het gezondheidsbevorderende effect tot stand brengen is een knelpunt voor het vertalen van de resultaten uit de epidemiologie naar de ontwikkeling van een functioneel voedingsingrediënt.De bescherming tegen kanker door groenten en fruit is toegeschreven aan antioxidanten zoals vitamine C, beta-caroteen en vitamine E, die op vele plaatsen in het lichaam, zoals de maag, darmen, long en de blaas actief zijn. Wetenschappelijk wordt momenteel veel aandacht besteed aan het mogelijke belang van andere belangrijke dieet componenten, zoals flavonoïden, als beschermende ingrediënten tegen ziekte. Flavonoïden komen in de natuur veel voor, en worden met name in hoge concentraties gevonden in fruit, groenten, knollen en zaden. Het gemiddelde Westerse dieet bevat ongeveer 1 gram aan flavonoïden per dag.Vele in vitro studies tonen aan dat flavonoïden goede antioxidanten en metaal chelatoren zijn. Daarnaast hebben ze anti-inflammatoire, anti-allergische en anti-virale eigenschappen die van belang worden geacht. Deze bevindingen verschaffen de basis voor de momenteel snel groeiende interesse om flavonoïden te gebruiken als functionele voedingsingrediënten. Als gevolg hiervan zou er in de nabije toekomst een toename in de opname van flavonoïden via het dieet verwacht kunnen worden. In winkels en via het internet worden voedingsmiddelen en voedingssupplementen gebaseerd op (iso)flavonoïden als functionele voedingsingrediënten verkocht. Dit, terwijl zowel de wetenschappelijke onderbouwing voor de gezondheidsclaims als gegevens die een gebalanceerde "risk-benefit" analyse mogelijk maken, nog ontbreken. In het geval van verhoogde toekomstige blootstelling van mensen aan flavonoïden worden voor de risk-benefit evaluatie vragen van belang rond hun mogelijk pro-oxidatieve chemisch gedrag. Er zijn aanwijzingen dat sommige flavonoïden mutageen zijn in zowel bacteriële als zoogdier in vitro test systemen. Een verhoogde mate aan maagkanker in bepaalde humane populaties is in verband gebracht met de consumptie van wijn met daarin mogelijk mutagene flavonoïden (Tamura et al. , Proc. Natl. Acad. Sci. USA. 77, 4961-4965, 1980, Hoey et al. , Am. J. Epidem., 113, 669-974, 1981). Alles samenvattend is er eigenlijk weinig bekend van de schadelijke maar ook van de gezondheidsbevorderende effecten van flavonoïden.Omdat flavonoid chinon/chinon methides genoemd zijn als de belangrijkste metabolieten die verantwoordelijk zouden zijn voor de mogelijke pro-oxidatieve toxiciteit en mutageniteit van flavonoïden, is karakterisering van deze pro-oxidant chemie van flavonoïden van belang. Echter er is weinig bekend over de structuur en de reactiviteit van deze flavonoid oxidatie producten. Daarom was het doel van deze studie de pro-oxidant chemie van flavonoïden te onderzoeken en een structuur-activiteits studie uit te voeren naar het chemische gedrag van 3',4'-dihydroxyflavonoïden. Daarbij werd speciale aandacht besteed aan de aard en reactiviteit van de gevormde chinon/chinon methide metabolieten. Met behulp van de GSH-trapping methode, HPLC, LC/MS, MALDI-TOF, 1H-NMR, 13C-NMR en kwantum-chemische computerberekeningen kon de chinon/chinon methide chemie van een serie 3',4'-dihydroxyflavonoiden gekarakteriseerd worden.De verkregen resultaten geven inzicht in de structuur-activteits relaties voor de pro-oxidatieve chemie van de electrofiele chinon /chinon methides metabolieten van de flavonoïden. De resultaten laten ook een onverwacht effect zien van de pH op het electrofiele gedrag van de B-ring catechol flavonoïden. Bovendien laten de resultaten van het proefschrift zien dat zelfs onder reducerende omstandigheden in een cellulair in vitro model (melanoma cellen) de pro-oxidatieve chemie van quercetine van belang kan zijn. Met name de vorming van glutathion-flavonoid conjugaten is een bewijs dat in het gekozen cellulaire model de pro-oxidatieve vorming van reactieve flavonoid chinon/ chinon methide metabolieten is opgetreden. Oxidatie van de catecholen naar chinonen en hun isomere chinon methides genereert electrofielen die DNA kunnen alkyleren. Van belang is dat de structurele randvoorwaarden die een flavonoid een goede antioxidant maken gelijk blijken te zijn aan de structurele kenmerken die essentieel zijn voor pro-oxidant gedrag en chinon methide vorming.Al met al is de pro-oxidant chemie van flavonoïden en van hun chinon /chinon methides verre van recht toe recht aan gebleken en zou de pro-oxidatieve chemie en de toxiciteit van de flavonoïden in het kader van hun gebruik als functional food ingredienten beter onderzocht en afgewogen moeten worden, rekening houdend met hun mogelijk gezondheidsbevorderende effecten

Genen voor je eten - eten voor je genen : maatschappelijke vragen en dilemma's rondom voedingsgenomics

In deze essyabundel wordt gezocht naar toepassingen van voedingsgenomics en naar maatschappelijke contexten waarin dat gebeurt. Met die kennis is het mogelijk om te bekijken welke maatschappelijke impact voedingsgenomics kan hebben op de productie en consumptie van voeding

Interactions of polyhalogenated aromatic hydrocarbons with thyroid hormone metabolism

This thesis deals with the possible interactions of polyhalogenated aromatic hydrocarbons and/or their metabolites with thyroid hormone metabolism. This chapter summarizes firstly the effects of thyroid hormone on the induction of biotransformation enzymes by PHAHs. Secondly, the results on the inhibition of thyroid hormone sulfation by hydroxylated metabolites of PHAH are summarized. Some conclusions and remarks on the overall implications of the results are given at the end of this chapter.The effects of thyroid hormone on the induction of biotransformation enzymes by polyhalogenated aromatic hydrocarbonsThe first part of this thesis focussed on the question whether or not the PHAH-induced decrease of plasma T4 is an adaptive endocrine response of the animal to cope with the onset of toxic effects by PHAHs. For this purpose, the possible regulatory effect of thyroid hormones on biotransformation enzymes was investigated, using rats differing in thyroid state which were exposed to TCDD or PCBs as model inducers of biotransformation enzymes.In Chapter 2 , the thyroid state of euthyroid (Eu), thyroidectomized (Tx) and Tx rats in which T3 or T4 levels are restored using osmotic minipumps were compared. The decreased circulatory levels of plasma T4 and T3, the increased pituitary feedback response (plasma TSH levels), as well as changed functional responses (decreased hepatic D1 and malic enzyme activities, and increased brain D2 activities) in Tx rats were largely restored to Eu levels in Tx+T4 rats and, except for plasma TT4 and brain D2 activity, in Tx+T3 rats. These results indicated that the thyroid hormone-replaced Tx rats were valid models to study peripheral effects of TCDD. Three days after exposure to 10 mg TCDD/kg body weight, plasma TT4 and FT4 levels were significantly reduced in Eu rats and in Tx+T4 rats, and plasma T3 was significantly reduced in Tx+T3 but not in Eu or Tx+T4 rats. Hepatic T4 UGT activity was induced by TCDD while T3 UGT activity was only slightly increased in the different exposed groups. These results strongly suggest that the thyroid hormone-decreasing effects of TCDD are predominantly extrathyroidal and mediated by the marked induction of hepatic T4 UGT activity.The effects of thyroid state modulation on the induction of detoxification enzymes by TCDD in experimental animals are described in Chapter 3 . In all rats, TCDD largely induced CYP1A1/1A2 activity (EROD), CYP1A1 protein content, and CYP1A1 mRNA levels. TCDD exposure also resulted in higher total hepatic cytochrome P450 content, hepatic p-nitrophenol UGT activity, and GST 1-1 protein levels, but had no effect on hepatic NADPH cytochrome P450 reductase activity, overall GST activity and GST 2-2, 3-3, and 4-4 protein levels and iodothyronine sulfotransferase activity. Thyroid state did not affect the total cytochrome P450, and GST activity and protein levels, but slightly decreased CYP1A1/2 activity, NADPH cytochrome P450 reductase activity, PNP UGT activity and iodothyronine sulfotransferase activity were demonstrated in Tx rats, as compared to Eu rats.In the second animal experiment, the interaction between thyroid state and PCBs in the regulation of CYP1A1 and CYP2B expression is described ( Chapter 4 ). Male Tx Sprague-Dawley rats, Eu rats, and rats made hyperthyroid by infusing T3 were treated with a single ip dose of the CYP2B inducer PCB 153 and the CYP1A inducer PCB 126. The thyroid states of the rats were confirmed by measurement of plasma T4, T3 and TSH and of functional parameters such as hepatic D1 activity, malic enzyme activity and a-glycerolphosphate dehydrogenase activity. Total hepatic cytochrome P450 content was increased by PCB treatment in all groups, but was not affected by thyroid state. NADPH cytochrome P450 reductase activity was decreased in Tx rats and increased in hyperthyroid rats, while PCB treatment had no effect. PCB 126 specifically induced T4 UGT activity, measured in the absence of detergent, and CYP1A activity, protein and mRNA levels, whereas PCB 153 induced T4 UGT activity, measured in the presence of the detergent Brij 56, and CYP2B activity, protein and mRNA levels. Thyroid state, neither hypo nor hyper, significantly affected T4 UGT activity or CYP1A and CYP2B activities, protein or mRNA levels.The almost complete lack of response of basal and PCB- or TCDD-induced activities of biotransformation enzymes to changes in thyroid state observed in our studies is in contrast to effects published by others (Kato and Takahashi et al. , 1968; Rumbaugh et al. , 1978; Leakey et al. , 1982; Müller et al. , 1983a/b; Skett, 1987; Yamazoe et al. , 1989; Arlotto and Parkinson, 1989; Murayama et al., 1991; Chowdhury et al. , 1983; Moscioni and Gartner, 1983; Pennington et al. , 1988; Goudonnet et al. , 1990; Williams et al. , 1986; Pimental et al. , 1993). This may be due to differences in strain and sex of the animals, the severity and duration of the hypo- and hyperthyroid states induced as well as the duration and dose of TCDD/PCB treatment. Overall, it can be concluded that hepatic NADPH cytochrome P450 reductase activity is dependent on thyroid state, whereas total cytochrome P450 as well as CYP1A1 and CYP2B together with UGT, GST and sulfotransferase activities show little or no thyroid hormone dependence. These slight effects are unlikely to represent an endocrine adaptation to a chemical stressor (TCDD). Therefore, the PHAH-induced decreased T4 levels , as well as other aspects of PHAH-induced alterations in thyroid hormone metabolism, are most likely a direct reflection of the developing toxicological response of the animals toward PHAH exposure.Inhibition of thyroid hormone sulfation by hydroxylated metabolites of polyhalogenated aromatic hydrocarbonsThe second part of this thesis focussed on the question whether or not hydroxylated metabolites of PHAHs (PHAH-OHs) are able to inhibit thyroid hormone sulfation in vitro as well as in vivo .Chapter 5 presents the investigations concerning the possible inhibitory effects of PHAH-OHs on iodothyronine sulfotransferase (SULT) activity. Rat liver cytosol was used as a source of sulfotransferase in an in vitro assay with 125I-labelled T2 as a model substrate. Hydroxylated metabolites of PCBs, PCDDs and PCDFs were found to be potent inhibitors of T2 SULT activity in vitro with IC50 values in the low micromolar range (0.2-3.8 mM). The most potent inhibitor of T2 SULT activity within our studies was the PCB metabolite 3-hydroxy-2,3',4,4',5-pentachlorobiphenyl with an IC50 value of 0.2 mM. A hydroxyl group in the para or meta position appeared to be an important structural requirement for T2 SULT inhibition by PCB metabolites. Ortho hydroxy PCBs were much less potent and none of the parent PHAHs were capable of inhibiting T2 SULT activity. In addition, the formation of T2 SULT-inhibiting metabolites from individual brominated diphenyl ethers and nitrofen as well as from some commercial PHAH mixtures (e.g. Bromkal, Clophen A50 and Aroclor 1254) by CYP450 catalyzed hydroxylation was also demonstrated.Consequently, the inhibition of thyroid hormone sulfation by PHAH-OHs was studied in more detail, investigating isozyme specificity and inhibition kinetics ( Chapter 6 ). The difference in inhibition pattern demonstrated for SULT activity present in rat liver and brain cytosol, is probably caused by a difference in isozyme pattern. It was shown that PCB-OHs inhibited T2 sulfation by interacting with the rat isozyme SULT1C1 and an additional isozyme responsible for T2 sulfation in female liver cytosol, probably rat SULT1B1, but not SULT1A1. On the other hand, human phenol SULT1A1 was inhibited by PCB-OHs, but not the human isozyme SULT1A3. In conclusion, we suggested that at least human SULT1A1, and rat SULT1C1 and perhaps rat SUL1B1 are involved in the inhibition of T2 sulfation by PCB-OHs. However, more information is needed about the various isozymes involved in iodothyronine sulfation in humans as well as in rats, before definite conclusions can be drawn.Furthermore, it is shown that T2 is a good model substrate for the active hormone T3 when investigating the inhibition of thyroid hormone sulfation by hydroxylated metabolites of PHAHs. The inhibition kinetics strongly suggested that the nature of the T2 sulfation inhibition by PCB-OHs is competitive. To obtain more decisive information, tests with purified isozymes should be performed. It was also demonstrated that PCDD-OHs and PCB-OHs themselves are substrates -albeit poor- for SULT enzymes, which further supports the competitive inhibition of thyroid hormone sulfation by PHAH-OHs.To bridge the gap between in vitro experiments using cytosol and the in vivo situation, we investigated the inhibition of thyroid hormone sulfation in hepatoma cell lines ( Chapter 7 ). Two PCB-OHs, 4-hydroxy-2',3,3',4',5-pentachlorobiphenyl and 4-hydroxy-3,3',4',5-tetrachlorobiphenyl, together with the known sulfation inhibitor pentachlorophenol (PCP) were tested in the rat hepatoma cell line FaO and the human hepatoma cell line HepG2. PCP inhibited T2 sulfation in vitro in FaO and HepG2 cells, although it was 1000 times less potent in whole cells than in rat liver cytosol. Micromolar concentrations of the two tested PCB-OHs hardly affected T2 conjugation in FaO cells, but reduced T2 sulfate formation in HepG2 cells. Inhibition of T2 sulfation was more pronounced using medium without FCS than in medium with 5% FCS, due to a lower uptake of inhibitor by the cells in the presence of serum, as demonstrated using radiolabeled PCP.These in vitro results indicate that hydroxylated PHAHs are potent inhibitors of thyroid hormone sulfation. Since thyroid hormone sulfation may play an important role in regulating "free" hormone levels in the fetus, and hydroxylated PCB metabolites are known to accumulate in fetal tissues after maternal exposure to PCBs, these observations in vitro might have implications for fetal thyroid hormone homeostasis and development.The in vivo experiment in which was tested if PHAH-OHs are able to inhibit T2 sulfation, was described in Chapter 8 . Pregnant rats were exposed to 25 mg Aroclor 1254/kg body weight or to the well-known phenol sulfation inhibitor PCP (25 mg/kg body weight) from day 10 till day 18 of gestation. Fetuses and dams were sacrificed on gestation day 20 (GD20). PCP and PCB metabolite levels in fetal serum and tissues were high. Aroclor 1254, but not PCP exposure resulted in an induction of hepatic EROD and T4 UGT activity in dams.PHAHs are known for their disrupting effects on thyroid hormone metabolism, as shown in Figure 9.1. In this animal experiment, Aroclor 1254 exposure caused an increase in T4 UGT activity, resulting in decreased TT4 levels. Treatment with PCP also resulted in decreased serum TT4 levels, but increased FT4 levels, in dams and fetuses. The ratio FT4/TT4 was increased indicating a reduced plasma TTR binding capacity in fetuses and dams following both treatments. D1 activity in liver decreased in dams and fetuses after treatment with Aroclor 1254 and PCP. This decrease is probably caused indirectly by the lowered T4 levels. D2 activity in brain decreased by exposure to PCP in dams but no effect was found in fetuses, and increased by exposure to Aroclor 1254 in fetuses, with no effect in dams. The increasing D2 activity is a response of the brain to low T4 levels, to maintain the T3 homeostasis.The positive control PCP was shown to increase the T2 SULT activity measured in maternal liver and brain cytosol. Studies using varying T2 concentrations and different protein concentrations suggested competitive inhibition of PCP carried over in the in vitro assay as well as true induction of T2 SULT activity. This effect of PCP on thyroid hormone sulfation in vivo apparently did not result in lower levels of the product T4S, since fetal and maternal serum levels of T4S were not changed after treatment with PCP. This negative answer may be explained by an increased availability of substrate (FT4; maternal) together with a reduced D1 activity by PCP treatment, resulting in a reduced enzymatic breakdown of T4S.Exposure to Aroclor 1254, which resulted in the formation of hydroxylated metabolites, did not significantly change the T2 SULT activity in maternal or fetal brain or liver cytosol, nor the serum levels of T4S.Remarkably, the T3S and T4S levels were very low in fetal rat serum in this study, especially when compared with the reported high iodothyronine sulfate levels in fetal human and sheep serum. This can not be explained by low SULT activity levels or high D1 activity levels in rat fetuses on day 20.Overall implications of the observed PHAH effects on thyroid hormone metabolismPHAHs induce a wide spectrum of toxic effects in rats. Some effects have been suggested to be linked to a hypothyroid situation, such as the "wasting syndrome", decreased feed intake, and increased cholesterol concentrations. Indeed, reduced serum T4 concentrations have been observed following exposure to PHAHs (Bastomsky et al. , 1977; Gorski and Rozman, 1987; Hermansky et al. , 1988; Brouwer, 1989; Beetstra et al. , 1991), and it is tempting to speculate about a relationship between the hypothyroxinemia and the observed toxic responses. However, induction of a hypothyroid situation or a hypothyroxinemia by PHAHs could also be regarded as an adaptive endocrine response to diminish the PHAH-induced toxicity. One argument in support of this interpretation is the observed protective effect of thyroidectomy on TCDD-induced lethality and immune toxicity (Rozman et al. , 1985).In this study, it is proposed that the T4 decrease could well have a regulatory role in the induction of hepatic biotransformation enzymes, as was reported before (see Chapter 1 ). The present investigations suggest that the lowering effects of PHAHs on T4 levels are only a toxic effect of PHAHs and not an adaptive response to regulate the induction of biotransformation enzymes. The differences with other reports on modulating effects of thyroid hormone state on biotransformation enzymes may be explained by differences in the time and dose of inducers as well as by a difference in hypo- or hyperthyroid state. Nevertheless, the T4 decreases in the hypothyroid animals in our study are similar to the PHAH-induced T4 decreases. Therefore, the model was good enough to investigate our hypothesis.The second part of this thesis demonstrated that the sulfotransferase enzyme is another thyroid hormone-binding protein, besides D1 and TTR, which can be competitively inhibited by hydroxylated metabolites of PHAHs. In a relatively narrow range of low micromolar concentrations, PHAH-OHs were able to competitively inhibit T2 SULT acttivity in vitro , in a SULT isozyme and tissue specific manner.Studies using a perinatal exposure setup were performed to test inhibition of T2 sulfation in vivo . It was demonstrated that the well-known sulfation inhibitor PCP was able to indeed competively inhibit T2 SULT activity, but also was able to upregulate the sulfotransferase protein amounts. Aroclor 1254 exposure resulted in a slight inhibition of T2 SULT activity, probably caused by hydroxylated metabolites formed. This inhibition, together with lower substrate (FT4) levels found after Aroclor treatment did not result in decreased serum T4S levels, which is probably caused by a concomitantly decreased inactivation route, i.e. a decreased D1 activity, together with a higher availibility of substrate (FT4) after PCP exposure.Remarkably, the serum T4S levels in fetal rat are low compared to the levels in sheep and human fetal serum samples (Wu et al. , 1992a/b; 1993a/b; Santini et al. , 1993). This could not be explained by already higher D1 activities or a relatively low sulfation activity in the control fetus around GD20. For this reason, we concluded that the fetal rat probably is not a very good model for humans in terms of investigating the impact of toxic compounds on fetal thyroid hormone sulfation. However, it should be mentioned that, although PHAHs and their metabolites interfere at many sites with thyroid hormone transport and metabolism, the fetus apparently is able to cope with those changes and can keep its homeostasis in T3.Another interesting point deduced from this study, is that PCP, which could be a model for PCB-OHs, itself showed effects on thyroid hormone levels and metabolism, indicating the importance of phenolic organohalogens compounds for disrupting effects on the thyroid hormone system. This also indicates that the disrupting effects of PCBs on the thyroid hormone system are for a large part caused by the hydroxylated metabolites formed. The own toxicity of PCB-OHs and related phenolic organohalogens inducing a separate set of effects together with the recently observed high fetal accumulation of hydroxy-PHAHs, give reason to further investigate the potential toxicity of these compounds on thyroid hormone metabolism and transport (see also Figure 9.1). It is worth mentioning that besides the "old" organohalogen pollutants that have been phased out since the 1980's, there is a wide range of new products on the market, such as brominated diphenylethers (PBDEs), chlorinated benzenes, bisphenol A and so on. PBDEs, which are nowadays used as flame retardants, have been demonstrated at increasing levels in our environment (De Boer et al. , 1989; Sellstrom et al. , 1996), and are probably able to cause similar effects as PHAHs. Serum T4 decreases have already been reported in rats after exposure to PBDEs (Darnerud et al. , 1996) or PCDEs (Rosiak et al. , 1997). Also, hydroxylated metabolites of PBDEs have been found to competitively inhibit the T4 binding to TTR in vitro (Meerts et al. , 1998).The human diet contains a diverse spectrum of naturally occuring and xeno-compounds that affect thyroid hormone metabolism. These include the organohalogens and related contaminants, and in addition, a large number of food components. Flavones and flavonoids have been reported to interfere with thyroid hormone binding proteins such as D1 (Auf'mkolk et al. , 1986; Cody et al. , 1989) and TTR (Lueprasitsakul et al. , 1990; Köhrle et al. , 1986). Flavonoids such as quercetin were similarly found to be able to inhibit phenol sulfotransferase activity in vitro (Walle et al. , 1995; Eaton et al. , 1996), and also other food additives were potent inhibitors of phenol sulfation (Bamforth et al. , 1993). The potential adverse human health impact of these compounds depends on a number of factors, including dietary intake, metabolism and pharmacokinetics, compound potency, serum concentrations, relative binding to serum proteins, and interactions or cross-talk with other endocrine pathways. In a risk evaluation, it should be taken into account that humans are exposed to a mixture of compounds with effects on thyroid hormone metabolism. If the mechanism of interference is similar for all these classes of compounds, the effects might very well be additive, or interactive. Additionally, the very persistent PHAHs are probably of more importance from a risk assessment point of view than the natural food components having a higher degradation rate.The effects of PHAHs on the thyroid hormone system in this study have been obtained in rats, are the results relevant for the human situation. Occupational or accidental exposure to high levels of PCBs or PBBs results in changes in serum T4 levels as was found by Bahn et al. (1980), Kreiss et al. (1982), Murai et al. (1987), and Emmet et al. (1988). Moreover, in pregnant women exposed to background levels of PHAHs mainly through diet, a significant negative correlation was observed between human milk levels of PHAHs and plasma T4 and T3 levels (Koopman-Esseboom et al. , 1994). In addition, increases in plasma TSH and both increases and decreases in plasma T4 levels were found in newborn babies following exposure to increasing PHAH levels through in utero and lactational transfer (Pluim et al. , 1993; Koopman-Esseboom et al. , 1994). Besides, prenatal exposure to PCBs is related to disorders in neurological development of children, found in some in epidemiologic studies (Rogan et al. , 1986; Jacobson et al. , 1990). It still is however not clear if these effects of PHAHs on thyroid hormone levels and metabolism may have possible effects on (brain) development.</p

Beslissen over biotechnologie

Environmen

Studie van de biologie en de kweek van zeebaars <i>Dicentrarchus labrax</i> (L.) in intensieve aquacultuurcondities

In Europe. the interest in sea bass (Dicentrarchus labrax) as a culture fish is fast increasing. This results in a rapid expansion of sea bass culture and of that of other marine fish species. such as seabream (Sparus aurata) and turbot (Scophtalmus maximus). In 1983 a research project was started, with the aim of culturing sea bass in the thermal effluents of the nuclear power station at Doel. In this way the possibility of producing valuable proteins is created while at the same time thermal effluents, normally dumped as waste heat, can be put to good use. The biology of the sea bass. In the Atlantic Ocean, the distribution area for sea bass ranges from Marocco to Norway. It is also found in the entire Mediterranean sea. The species has only one reproductive period, independent of the latitude. Spawning usually occurs in the littoral zone, in water with a salinity of above 30 promille. The juvenile stages, however, are mostly found in brackish, shallow waters, which function as "nursery grounds" due to their rich food supplies. This food mainly consists of invertebrates and, although some adult piscivore sea bass can be found, crustaceans remain the most important food items. Sea bass are very euryhaline and eurytherm, which has been experimentally determined and which can also be concluded from their local and geographical distribution. Sea bass as culture fish. Although sea bass culture only became popular in the eighties, a strong interest in the technique has developed owing to the high quality of the meat yielded and its subsequent high market value. The extensive culture is mainly situated in Italy where, in the "'allicultura", 700 tons are harvested yearly. However, while during cold winter months mortality is high, the yield per surface unit in extensive culture is limited. For this reason nowadays more interest, research and money are invested in the intensive culture system where densities up to 60 kg/m² are obtained. Besides the less applied pond culture, net cages and tanks are mainly used in intensive sea bass culture, depending on the suitability of the site. Both culture methods have advantages and disadvantages. Culture in tanks allows a better control of the fish population and the output per m² is higher than that in cages. However, financial investments for cage culture are much lower when compared to tank culture. In Europe the most important sea bass producing countries are France, Italy, Spain, Greece and Yougoslavia. Together they produce about 6000 tons of sea bass and sea bream per year. The high prices which can rise to 600 Bfr/kg, and which can show a yearly increase of about 20 %, are expected to stabilize in the near future. A decrease, however, is not to be expected because supply of fry is very limited and prevents a fast increase of production. A direct consequence of the shortage of juveniles is their high price, which rises to above 35 Bfr/piece. The pilot plant at Doel. The first culture experiments with sea bass in our country date from 1982 and were performed at the Zoological Institute of the K.U.Leuven. Due to a number of problems inherent to the recirculation system, which was used in this experiment, we started in 1983 by building a pilot installation for the culture of sea bass and eel on the K.C.D. grounds. When this installation was completed in 1985, the total effective water surface was 90 m², divided over 4 round and 6 square tanks. The water supply is derived from the River Scheldt, from the cooling tower and from the condenser circulation, in order to optimize the temperature and oxygen concentration of the culture water in the tanks throughout the year. In the tanks, the water is renewed once an hour; it leaves the tank through a central exit, which is constructed in such a way that, during the cleaning process, all the silt deposit is removed. Each tank is provided with extra aeration and an automatic feeding system (EWOS Multic feeder). The water tempe