Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Aerial surveys of cetaceans and seabirds in Irish waters : occurrence, distribution and abundance in 2015-2017
Rogan, E. ; Breen, P. ; Mackey, Mick ; Cañadas, Ana ; Scheidat, M. ; Geelhoed, S.C.V. ; Jessopp, Mark - \ 2018
- 298 p.
observe programme - aerial survey - cetacean - seabird - abundance - density - megafauna - distance sampling - Ireland - Atlantic - Celtic Sea - Irish Sea
New insights into ocean sunfish (Mola mola) abundance and seasonal distribution in the northeast Atlantic
Breen, Patricia ; Cañadas, Ana ; Ó Cadhla, Oliver ; Mackey, Mick ; Scheidat, Meike ; Geelhoed, Steve C.V. ; Rogan, Emer ; Jessopp, Mark - \ 2017
Scientific Reports 7 (2017). - ISSN 2045-2322 - 9 p.
The ocean sunfish, Mola mola, is the largest teleost fish in the world. Despite being found in all oceans of the world, little is known about its abundance and factors driving its distribution. In this study we provide the first abundance estimates for sunfish in offshore waters in the northeast Atlantic and the first record of extensive sunfish presence in these waters year-round. Abundance estimates and predictive distributions for sunfish in approximately 300,000 km² of the northeast Atlantic were derived from large scale offshore aerial surveys in 2015–2016 using distance sampling techniques. Generalized additive models of sunfish density were fitted to survey data from 17,360 km of line transect effort resulting in minimum abundance estimates of 12,702 (CI: 9,864-16,357) in the summer (Density = 0.043 ind/km²) and 8,223 individuals (CI: 6,178-10,946) (Density = 0.028 ind/km²) in the winter. Density surface models predicted seasonal shifts in distribution and highlighted the importance of the mixed layer depth, possibly related to thermoregulation following deep foraging dives. The abundance estimate and estimated daily consumption of 2,600 tonnes of jellyfish in the northeast Atlantic highlights the need to re-assess the importance of this species in the pelagic ecosystem, and its role in top-down control of jellyfish blooms.
Phylogeography and population dynamics of the white-sided dolphin (Lagenorhynchus acutus) in the North Atlantic
Banguera-Hinestroza, E. ; Evans, P.G.H. ; Mirimin, L. ; Reid, R.J. ; Mikkelsen, B. ; Couperus, A.S. ; Deaville, R. ; Rogan, E. ; Hoelzel, A.R. - \ 2014
Conservation Genetics 15 (2014)4. - ISSN 1566-0621 - p. 789 - 802.
Highly mobile species in the marine environment may be expected to show little differentiation at the population level, but this is often not the case. Instead cryptic population structure is common, and effective conservation will require an understanding of how these patterns evolve. Here we present an assessment from both sides of the North Atlantic of differentiation among populations of a dolphin species that inhabits mainly pelagic waters, the Atlantic white-sided dolphin. We compare eleven putative populations in the western and eastern North Atlantic at mtDNA and microsatellite DNA loci and find reduced nucleotide diversity and signals for historical bottlenecks and post-bottleneck expansions in all regions. We calculate expansion times to have occurred during the early Holocene, following the last glacial maximum (LGM). We find evidence for connectivity among populations from either side of the North Atlantic, and differentiation between putative populations in the far northeast compared with all other areas sampled. Some data suggest the possibility of separate refugia during the LGM explaining this pattern, although ongoing ecological processes may also be a factor. We discuss the implications for developing effective programs of conservation and management in the context of ongoing anthropogenic impact.
An interactional approach to framing in conflict and negotiation
Dewulf, A. ; Gray, B. ; Putnam, L. ; Bouwen, R. - \ 2011
In: Framing matters. Perspectives on negotiation research and practice in communication / Donohue, W.A., Rogan, R.G., Kaufman, S., New York : Peter Lang - ISBN 9781433111495 - p. 7 - 33.
Competing Claims in Public Space: The Construction of Frames in Different Relational Contexts
Aarts, N. ; Lieshout, M. van; Woerkum, C. van - \ 2011
In: Framing Matters. Perspectives on Negotiation Research and Practice in Communication / Rogan, R.G., Donohue, W.A., Kaufman, S., New York : Peter Lang - ISBN 9781433111495 - p. 234 - 254.
Framing Nature Conservation Experts and Expertise in the Drentsche Aa Area in the Netherlands: A Contextual Approach
Bommel, S. van; Aarts, N. - \ 2011
In: Framing Matters. Perspectives on Negotiation Research and Practice in Communication / Donohue, W.A., Rogan, R.G., Kaufman, S., New York : Peter Lang - ISBN 9781433111495 - p. 191 - 209.
The Future Today
Rothman, D.S. ; Agard, J. ; Alcamo, J. ; Alder, J. ; Al-Zubari, W.K. ; Beek, T. aus der; Chenje, M. ; Eickhout, B. ; Flörke, M. ; Galt, M. ; Ghosh, N. ; Hemmings, A. ; Hernandez-Pedresa, G. ; Hijioka, Y. ; Hughes, B. ; Hunsberger, C. ; Kainuma, M. ; Kartha, S. ; Miles, L. ; Msangi, S. ; Odongo Ochola, W. ; Pichs Madruga, R. ; Pirc-Velkarvh, A. ; Ribeiro, T. ; Ringler, C. ; Rogan-Finnemore, M. ; Sall, A. ; Schaldach, R. ; Stanners, D. ; Sydnor, M. ; Ruijven, B. van; Vuuren, D. van; Verburg, P.H. ; Verzano, K. ; Zöckler, C. - \ 2007
In: United Nations Environment Programme, Global Environment Outlook GEO 4 Environment for Development (Section E - The Outlook - Towards 2015 and Beyond, Chapter 9) / Magadza, C., Nairobi, Kenya : UNEP - ISBN 9789280728361 - p. 397 - 454.
Modelling discard ogives from Irish demersal fisheries
Borges, L. ; Zuur, A.F. ; Rogan, E.G. ; Officer, R. - \ 2006
ICES Journal of Marine Science 63 (2006)6. - ISSN 1054-3139 - p. 1086 - 1095.
sea - gadoids
Annual discard ogives were estimated using generalized additive models (GAMs) for four demersal fish species: whiting, haddock, megrim, and plaice. The analysis was based on data collected on board commercial vessels and at Irish fishing ports from 1995 to 2003. For all species the most important factors influencing annual discard ogives were fleet (combination of gear, fishing ground, and targeted species), mean length of the catch and year, and, for megrim, also minimum landing size. The length at which fish are discarded has increased since 2000 for haddock, whiting, and plaice. In contrast, discarded length has decreased for megrim, accompanying a reduction in minimum landing size in 2000
Fluorobenzo[a]pyrenes as probes of the mechanism of cytochrome P450-catalyzed oxygen transfer in aromatic oxygenations
Mulder, P.P.J. ; Devanesan, P. ; Alem, K. van; Lodder, G. ; Rogan, E.G. ; Cavalieri, E.L. - \ 2003
Free Radical Biology and Medicine 34 (2003)6. - ISSN 0891-5849 - p. 734 - 745.
one-electron oxidation - rat-liver microsomes - radical cations - horseradish-peroxidase - semiempirical methods - mouse skin - benzo<a>pyrene - dna - identification - metabolism
Fluoro substitution of benzo[a]pyrene (BP) has been very useful in determining the mechanism of cytochrome P450-catalyzed oxygen transfer in the formation of 6-hydroxyBP (6-OHBP) and its resulting BP 1,6-, 3,6-, and 6,12-diones. We report here the metabolism of 1-FBP and 3-FBP, and PM3 calculations of charge densities and bond orders in the neutral molecules and radical cations of BP, 1-FBP, 3-FBP, and 6-FBP, to determine the mechanism of oxygen transfer for the formation of BP metabolites. 1-FBP and 3-FBP were metabolized by rat liver microsomes. The products were analyzed by HPLC and identified by NMR. Formation of BP 1,6-dione and BP 3,6-dione from 1-FBP and 3-FBP, respectively, can only occur by removal of the fluoro ion from C-1 and C-3, respectively, via one-electron oxidation of the substrate. The combined metabolic and theoretical studies reveal the mechanism of oxygen transfer in the P450-catalyzed formation of BP metabolites. Initial abstraction of a ¿ electron from BP by the [Fe4+=O]+¿ of cytochrome P450 affords BP+¿. This is followed by oxygen transfer to the most electropositive carbon atoms, C-6, C-1, and C-3, with formation of 6-OHBP (and its quinones), 1-OHBP, and 3-OHBP, respectively, or the most electropositive 4,5-, 7,8-, and 9,10- double bonds, with formation of BP 4,5-, 7,8-, or 9,10-oxide.
Interactions of polyhalogenated aromatic hydrocarbons with thyroid hormone metabolism
Schuur, A.G. - \ 1998
Agricultural University. Promotor(en): P.J. van Bladeren; T.J. Visser; A. Brouwer. - S.l. : Schuur - ISBN 9789054859406 - 173
gechloreerde koolwaterstoffen - schildklierhormonen - chlorinated hydrocarbons - thyroid hormones

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 hydrocarbons
The 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 hydrocarbons
The 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 metabolism
PHAHs 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.

Synthesis and structure determination of the adducts formed by electrochemical oxidation of 1,2,3,4-tetrahydro-7,12-dimethyl benz[a]anthracene in the presence of deoxyribonucleosides or adenine
Mulder, P.P.J. ; Cheng, L. ; Sekhar, B.C. ; George, M. ; Gross, M.L. ; Rogan, E.G. ; Cavalieri, E.L. - \ 1996
Chemical Research in Toxicology 9 (1996)8. - ISSN 0893-228X - p. 1264 - 1277.
Polychlorinated biphenyl-induced alterations of thyroid hormone homeostasis and brain development in the rat
Morse, D.C. - \ 1995
Agricultural University. Promotor(en): J.H. Koeman; A. Brouwer. - S.l. : Morse - ISBN 9789054853756 - 175
toxische stoffen - polychloorbifenylen - zenuwstelsel - zintuigorganen - neurofysiologie - neurologie - osmose - tropismen - schildklier - ratten - xenobiotica - fysische factoren - chemische factoren - toxic substances - polychlorinated biphenyls - nervous system - sense organs - neurophysiology - neurology - osmosis - tropisms - thyroid gland - rats - xenobiotics - physical factors - chemical factors - cum laude
Introduction

The work described in this thesis was undertaken to gain insight in the processes involved in the developmental neurotoxicity of polychlorinated biphenyls. It has been previously hypothesized that the alteration of thyroid hormone status by PCBs may be in part responsible for the developmental neurotoxicity of these compounds in humans (Rogan et al. 1986). This is a logical hypothesis, given the well-described effects of PCBs on plasma thyroid hormone levels in adult animals, and the indisputable importance of thyroid hormones in brain development.

Therefore the first goal was to determine the nature and mechanism of PCBinduced decreases in circulating and brain thyroid hormone levels in fetal and neonatal rats (Chapter 2,3,4 and 5). We examined the effects of maternal PCB administration on the metabolism of thyroid hormone in the brain and liver of fetal, neonatal and adult offspring in relation to the level of thyroid hormone in the plasma and brain. Vitamin A status, which may be linked to thyroid hormone status following PCB exposure, was also examined in one reproduction study (Chapter 6).

Since the kinetics and metabolism of PCBs may play a pivotal role in the alteration of thyroid hormones, a radiolabelled and easily metabolized PCB congener was used in in vivo (Chapter 2) and in vitro experiments (Chapter 3) to examine the kinetics and metabolism of a model compound in pregnant and fetal rats. The relevance of this model compound for complex PCB mixtures was ascertained following the administration of a commercial PCB mixture to pregnant rats (Chapter 5).

Lastly, neurochemical analysis were conducted on the brains of the offspring following maternal PCB exposure to determine which brain regions, cell types and neurotransmitter systems are affected during brain development (Chapter 7 and 8).

Biotransformation of PCBs to thyroid hormone antagonists

The metabolism and distribution of [ 14C]-3,3',4,4'-tetrachlorobiphenyI ([ 14C]- TCB) was examined in pregnant rats and their fetuses (Chapter 2 and 3, Morse et al., 1995). The major metabolite found in adult liver and plasma, placental tissue, whole fetuses and fetal blood was 3,3',4',5-tetrachloro-4-biphenyloI (4-OH-tetraCB). While maternal tissue levels of [ 14C]-TCB derived radioactivity significantly decreased by 65-85% over a 7 day period, radioactivity in the fetus accumulated more than 100-fold over the same period. The fetal accumulation of radioactivity was due primarily to 4-OH-tetraCB, and on day 20 of gestation, fetal plasma levels of 4-OH- tetraCB were 14 times higher than maternal plasma levels (14 μM vs 1 μM).

In order to determine the source of 4-OH-tetraCB in the fetus, in vitro studies were carried out by incubating [ 14C]-TCB with maternal and fetal rat microsomes and analysing the reaction products with high pressure liquid chromatography and gas chromatographic/mass spectrometric analysis. First, incubation conditions were optimized using male rat microsomes. Under optimal incubation conditions, hepatic microsomes from pregnant rats pretreated with TCB produced 4-OH-tetraCB as the major metabolite, while no metabolites were detected in incubations with microsomes from fetuses from pregnant rats pretreated with TCB. The results indicate that 4-OH-tetraCB, found in the fetal compartment is due to transplacental transport from maternally formed 4-OH-tetraCB. This is in agreement with the observation that the biotransformation of TCB is dependent on CYP1A1 induction, and no CYP1A1 activity was observed in fetal microsomes after maternal treatment with TCB.

In late gestation, the high levels of 4-OH-tetraCB found in the fetal plasma were asssociated with decreases in fetal plasma thyroid hormone levels in the absence of significant decreases in maternal plasma thyroid hormones. 4-OH-tetraCB has a high affinity for transthyretin (the major plasma thyroid hormone transport protein in the rat) and competitively displaces thyroxine from this protein (Brouwer et al. 1990, Lans et al. 1993). It was therefore concluded that the accumulation of 4-OH-tetraCB in the fetus is due to the high affinity of this metabolite for transthyretin, and results in significant decreases in fetal plasma thyroxine levels.

Since the model compound 3,3',4,4'-TCB is present in only very low levels in the environment, it was of interest if the exposure of pregnant rats to a commercial PCB mixture (Aroclor 1254) would also result in the accumulation of phenolic metabolites in the fetal plasma (Chapter 5). Relatively high levels of hydroxylated PCB metabolites from penta, hexa. and hepta- chlorinated biphenyls have been found in the plasma of rats exposed to Aroclor 1254 and in environmentally exposed humans (Bergman et al. 1994). A significant accumulation of 4-OH- 2,3,3',4',5-pentachlorobiphenyI (4-OH-pentaCB) was found in the plasma of late gestational fetuses from pregnant rats exposed to Aroclor 1254 (up to 4.6 μM). This PCB metabolite has a 10-fold higher binding affinity for TTR than thyroxine, thereby confirming the relevance of work with the model compound, 3,3',4,4'-tetrachlorobiphenyl. In addition, relatively large amounts of 4-OH- pentaCB were found in the fetal (0.46 μM), but not weanling rat brain, indicating that in the absence of a functional blood-brain barrier hydroxylated PCB metabolites may enter the brain. The toxicological significance of this finding deserves investigation.

Effects of PCB exposure on thyroid homone levels and metabolism

T 4 -Uridine-diphospho-glucuronyl transferase
Decreases in plasma thyroid hormone levels in adult rodents may also be caused by the induction of the hepatic glucuronidation of thyroxine (Bastomsky, 1974, Barter and Klaasen 1992). The effect of a single maternal dose of 3,3',4,4',5,5'-hexachlorobiphenyl (HCB) on day 1 of gestation and in combination with repeated maternal doses of TCB (day 2 to 18 of gestation) on maternal, fetal and neonatal hepatic microsomal and brain thyroxine metabolism is described in Chapter 4. The results indicated that although maternal administration of coplanar PCBs may result in the induction of fetal hepatic microsomal T 4 glucuronidation, this induction did not cause the reductions in fetal plasma T 4 levels. Only the combined dose of HCB with TCB resulted in significant decreases in fetal plasma T 4 levels. This indicates that decreased placental transport of maternally-derived T 4 and the blockage of fetal thyroid hormone transport by 4-OH-tetraCB resulted in the decrease of fetal T 4 levels. In neonates and dams, however, the induction of T 4 glucuronidation by lactational exposure to coplanar PCBs may contribute to the observed decreases in plasma thyroxine levels.

Maternal exposure to the commercial PCB mixture Aroclor 1254 also induced T 4 -UDPGT activity in hepatic microsomes from pregnant and weanling rats, but not in the fetus (Chapter 5). Since only the induction of maternal hepatic microsomal T 4 -UDPGT correlated with reductions in plasma thyroid hormones, it was concluded that the induction of T4-UDPGT activity played only a minor role in the reductions of plasma thyroid hormones in fetal and weanling rats. Large reductions in plasma thyroid hormones have also been observed following dietary Aroclor 1254 exposure in homozygous Gunn rats, which are deficient in T 4 -UDPGT activity (Collins and Capen, 1980a). The only long-term effect on thyroid hormone metabolism observed following maternal PCB exposure was a significant decrease in female hepatic microsomal T 4 glucuronidation in young adult offspring.

Type II thyroxine 5'-deiodinase
As most of the biologically active hormone triiodothyronine (T 3 ) is derived from T 4 by deiodination in the brain by Type II thyroxine 5'-deiodinase 5'D-II Silva and Larsen, 1982, Kaplan et al. 1983), it was of interest to examine the effects of PCBinduced reductions in plasma T 4 levels on 5'D-II activity. Decreases in brain T 4 levels result in a slower turnover of the enzyme, yielding a higher activity per unit protein in brain homogenates (Leonard et al. 1984). This regulatory mechanism is important in maintaining brain T 3 levels. In Chapter 4, the significant decreases in fetal, neonatal and weanling rat plasma T 4 levels following coplanar PCB exposure were accompanied by significant increases in 5'D-II activity in brain homogenates. It was concluded that the increases in 5'D-II activity were in compensation for low T 4 levels in the developing rat brain, which could be detrimental for normal brain development if insufficient T 3 was formed from T 4 .

Following maternal exposure to Aroclor 1254, reductions in fetal plasma T 4 were also accompanied by increases in brain 5'D-II activity. However, in contrast to the effects observed with coplanar PCBs in weanling rats, 5'D-II activity was decreased in weanling rats with normal plasma and brain T 4 levels, and equal to control values when plasma and brain T, levels were decreased. This can not be explained by the current knowledge of 5'D-II regulation.

Plasma and brain thyroid hormone levels
In the current study, the effect of maternal PCB exposure on plasma thyroid hormone levels was transient, with only mild effects observed in weanling rats. Despite the significant lactational transfer of PCBs to the neonate, the effects on neonatal thyroid hormone homeostasis are less severe in neonates as in the fetus. Several mechanisms appear to be involved that may explain the difference in responses between fetuses and weanling rats: the induction of maternal hepatic T 4 glucuronidation late in gestation, the accumulation of hydroxylated PCB metabolites in the fetus, and reduced placental transfer of T 4 . Also the dilution of the tissue PCB levels during postnatal growth and the fecal and urinary excretion of PCBs may reduce the severity of plasma T 4 reductions in weanling rats following gestational PCB exposure. For example, the continuous postnatal dietary exposure of maternal rats to Aroclor 1254 results in low plasma T 4 levels throughout the weaning period (Collins and Capen, 1980b).

Despite severe decreases in fetal plasma and brain T 4 levels following maternal PCB exposure, only marginal decreases were observed in fetal brain T 3 levels. This indicates that the late gestational rat fetus can maintain brain T 3 levels by an increase in 5'D-II activity, and is at little risk for PCB-induced hypothyroidism, at least in the brain.

The observation that plasma TSH levels did not increase following PCB-induced decreases in plasma T 4 levels in the fetus and plasma T 3 and T 4 levels in the neonate suggests that the developing brain may have been euthyroid. However, the decreases in plasma T 4 levels themselves could be expected to result in an increase in TSH levels. Similar decreases in plasma T 4 levels in late gestational fetal Wistar rats following maternal treatment with methimazole have been shown to result in an 600% increase in plasma TSH levels (Morreale de Escobar et al. 1993), and it is likely that fetal TSH levels are modulated predominately by plasma T 4 rather than T 3 . In adult rats, significant increases in plasma TSH levels have been observed following dietary exposure to Aroclor 1254 that resulted in that decreases in plasma T 4 , but not T 3 at the same time point (Barter and Klaassen, 1994). A weak effect of PCBs on TSH secretion has been observed following a relatively high dietary exposure to Aroclor 1254, after which the rise in plasma TSH was suprisingly low in comparison to the rise in plasma TSH following dietary exposure to polychlorinated naphthalenes, which induced similar decreases in plasma T 4 levels as PCBs (Barter and Klaassen, 1994).

In conclusion, maternal. PCB exposure during gestation results in a large decrease of fetal brain T 4 levels, but only marginal decreases in T 3 levels in the late gestational rat fetus. It is possible that earlier in gestation, before 5'D-II activity can compensate for decreases in brain T 4 levels, significant reductions in brain T 3 levels are induced by maternal PCB treatment.

Retinoids

Analogous to thyroid hormones, retinoids play a crucial role in brain development, although their most important effects are during early and mid-gestation (Adams, 1993). To evaluate retinoid status, plasma and hepatic retinol and retinylesters were determined following maternal Aroclor 1254 exposure. The reductions in plasma retinol levels may be caused by the accumulation of 4-OH- pentaCB; in the plasma, analogous to the disruption of the binding of retinol binding protein to transthyretin by 4-OH-tetraCB following exposure to 3,3',4,4'-tetrachlorobiphenyl. Although the effects of maternal PCB exposure on retinoid homeostasis in the fetus, neonate and young adult offspring appear to be minor, the regulation of retinoid homeostasis exhibited long-term alterations in the PCB exposed group.

Alterations in neurochemistry

In Chapter 8 and 9 the effects of maternal PCB (Aroclor 1254) exposure were examined on the ontogeny of biogenic amines, a glial cell marker (glial fibrillary acidic protein, GFAP) and a neuronal cell marker (synaptophysin) in diverse brain regions.

Biogenic amines
Of the biogenic amines examined, only the levels of 5-hydroxytryptamine (5-HT, serotonin) and its metabolite, 5-hydroxy-indoleacetic acid (5-HIAA) were altered by pre- and postnatal PCB exposure. It is notable that in adult animals the dopaminergic system is the most sensitive for exposure to commercial PCB mixtures, while we found no effects on the levels of dopamine or its major metabolite in the brains of PCB-exposed offspring (Seegal et al. 1985, 1986a, 1986b, 1991). Pre- and postnatal exposure to the lightly chlorinated PCB mixture Aroclor 1016 resulted in transient increases in striatal dopamine levels (Seegal, 1994). Therefore the effects of PCB exposure on regional brain monoamine metabolism during development do not resemble the effects in adult animals.

In general, the effects can be characterized by an increase in 5-HIAA concentrations and the 5-HIAA/5-HT ratio in the lateral olfactory tract and prefrontal cortex, and an increase in 5-HIAA levels in the striatum and hippocampus on postnatal day 90. Since the effects on the serotonergic system are almost absent on day 21 postpartum when exposure to PCBs via lactation ceased, there appears to be a delayed effect on the ontogeny of serotonin metabolism.

GFAP and Synaptophysin levels
The most consistent effects of maternal PCB exposure on GFAP levels were observed in the lateral olfactory tract and the brainstem. Increases in GFAP concentrations were observed in both male and female offspring 21 and 90 days after birth. Increases were also observed in cerebellar GFAP levels on 21 and 90 days postpartum. In the brainstem of male and female offspring maternal PCB exposure prevented the increase in GFAP concentrations that was observed in control offspring, indicating a delay in the ontogeny of brainstem GFAP expression.

Synaptophysin levels in the brain of the offspring were affected in a more complex manner than GFAP following maternal PCB exposure. Following maternal PCB exposure, the most sensitive brain regions from both sexes for decreases in synaptophysin concentrations on postnatal day 21 were the lateral olfactory tract and the brainstem. However, in young adult animals brainstem synaptophysin levels were significantly decreased in males and significantly increased in females. Synaptophysin levels were also significantly decreased in the striatum and hypothalamus of female, but not male offspring following maternal PCB exposure.

The mechanisms involved in the alterations of GFAP and synaptophysin levels in the brains of the PCB-exposed offspring are not yet been elucidated. Increases in GFAP levels accompanied by decreases in synaptophysin levels in the lateral olfactory tract and prefrontal cortex are characteristic of reactive gliosis following neuronal loss (O'Callaghan and Miller, 1989). The decreases in GFAP and synaptophysin levels in the brainstem in weanling rats may be indicative of a developmental delay in brainstem maturation. The raphe nuclei in the brainstem contain serotonergic neurons which project to the lateral olfactory tract and the prefrontal cortex (Kosofsky and Molliver, 1987). It is therfore conceivable that the alterations in serotonergic matabolism as well as GFAP and synaptophysin levels in the lateral olfactory tract and prefrontal cortex result from a developmental delay in the serotonergic innervation of these brain regions.

Relevance of the conducted research for human development and future toxicological research

Thyroid hormones
In a recently published study of 105 mother-infant pairs, elevated maternal body burdens of polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls (levels in milk fat) were shown to be associated with alterations of human thyroid hormone status (Koopman-Esseboom et al. 1994). The effects are characterized as negative correlation of PCDD, PCDF and PCB congeners with maternal plasma TT 3 before delivery and maternal plasma TT 4 and TT 3 after delivery, and a positive correlation with plasma TSH levels in the infants in the second week and third month after birth. In infants with a higher exposure, plasma TT 4 levels were significantly lower (10%) and TSH levels were significantly higher (37%). Maternal body burden of three PCB congeners (CB 118, CB 138 and CB 153) was positively correlated with umbilical plasma TSH levels. In a similar study with 38 mother-infant pairs an increase in infant plasma TSH levels and plasma TT 4 levels was observed in infants with a higher exposure to the total toxic equivalents of PCDDs and PCDFs (Pluim et al., 1992).

The relative effects of PCBs, PCDFs and PCBs on plasma thyroid hormone levels observed by Koopman-Esseboom et al. (1994) in mother-infant pairs are generally the same as observed in adult mice, rats and monkeys at much higher doses. In contrast to the decreases observed in late gestational fetal rat plasma TT 4 and FT 4 following maternal exposure to Aroclor 1254, no effect was observed of maternal body burden on umbilical cord TT 4 and FT 4 levels.

It is unlikely that the alterations in thyroid hormone homeostasis associated with maternal PCB and PCDD levels observed in human newborns and infants will result in developmental alterations of the nervous system. Compensatory mechanisms, such as the induction of Type II 5'- thyroxine deiodinase in the brain, should offset decreases in plasma and brain T 4 levels. In the late gestational rat, near normal brain T 3 levels were maintained despite severe decreases in plasma and brain T 4 levels following maternal PCB exposure. However, the potential exists for significant reductions in fetal thyroid hormone levels earlier in gestation before compensatory mechanisms have fully developed or in specific brain regions not examined in this study.

There are several important aspects in which the thyroid hormone transport differs between humans and rats with possible consequences for the effects of PCBs. The main thyroid hormone transport protein in humans is TBG, while in rats TTR is the major protein (Robbins, 1991). Although under certain circumstances TBG may be present in rats, it has a low affinity for T 4 (Rouaze-Romet et al. 1992). Hydroxylated PCB metabolites bind only very weakly to TBG, so it is possible that the impact of hydroxylated PCB metabolites on plasma T 4 levels in humans may be minor. However, the fetal mouse has both TTR and TBG as transport proteins, and mouse TBG has similar binding properties to human TBG (Vrancks et al. 1990), so the mouse may be a better model than the rat for studying the effects of PCBs on thyroid hormone transport. Recent research has shown that following maternal TCB exposure 4-OH-tetraCB accumulates in the fetal mouse, binds to TTR, resulting in the decrease of fetal plasma T 4 levels (unpublished results, D.C. Morse and P.O. Darnerud). Therefore, it is possible that transplacental. transport of hydroxylated PCBs in humans results in the decrease plasma and brain thyroid hormone levels before the rise of fetal hypothalamic-pituitary function in mid-gestation.

While it is generally accepted that thyroid hormone deficiency in neonates and in late gestation has a negative effect on brain development in the rat as well as humans, the effects of thyroid hormone deficiency earlier in gestation are not clearly understood (Morreale de Escobar et al. 1993, review, Porterfield and Hendrich, 1993, review). Thyroid hormone and their receptors have been found in human fetuses by 10 weeks of gestation (Fisher, 1985), although the functional significance of these observations is currently unclear. The finding of Pharoah et al. (1972) that neurological damage of endemic cretinism could be prevented if iodized oil was given to the mother before the second trimester of pregnancy supports a role of thyroid hormones in brain development in this period.

Therefore several questions remain to be answered: does maternal PCB exposure result in significant decreases in brain thyroid hormones in early and mid-gestation in rodents and humans, and whether such decreases are relevant for brain development.

Effects on neurochemical development
Gestational and lactational exposure to a commercial PCB mixture, Aroclor 1254 resulted in long-term effects on the neurochemical development of the progeny of rats (Chapter 7 and 8). The study does not support the hypothesis that PCB-induced pre- or postnatal hypothyroidism was the cause of the neurochemical alterations (Chapter 5). The study does give an indication which neurotransmitter systems, which cell types and which brain areas may be affected by in utero and lactational exposure to a higher chlorinated PCB mixture, providing a solid base for further research.

One of the questions which has interested researchers in PCB-induced toxicity for nearly 20 years is which PCB congeners are responsible for the toxicity of these compounds. This question has only been adequately answered for the immunotoxicity and some developmental endpoints (teratogenesis and fetotoxicity) in which the interaction of the PCB congeners with the Ah-receptor plays an important role.

To date, reports have been published on the behavioral neurotoxicity of only two individual PCB congeners, 3,3',4,4'-tetrachlorobiphenyl and 3,3',4,4',5-pentachlorobiphenyl, both of which are coplanar PCBs with a high affinity for the Alireceptor. 3,3',4,4'-tetrachlorbiphenyl was shown to be a developmental neurotoxin in mice following high dose gestational exposure, reducing striatal dopamine and dopamine receptor levels in mice, delaying advoidance behavior and inducing neuropathological alterations in the cranial roots (Tilson et al. 1979, Chou et al. 1979, Agrawal et al. 1981). Postnatal exposure of mice to 3,3',4,4'-tetrachlorobiphenyl has also been shown to affect hippocampal muscarinic receptor levels and alter spontaneous activity (Eriksson, 1988, Eriksson et al. 1991). Due to the rapid metabolism of 3,3',4,4'-tetrachlorobiphenyl and accumulation of hydroxylated metabolites in the fetus no conclusions can be drawn as to the role of the parent compound or its metabolites in the developmental neurotoxicity of this PCB congener (Morse et al. 1995).

On the other hand, exposure of pregnant rats to poorly metabolizable 3,3',4,4',5- pentachlorobiphenyl, delayed the onset of spontaneous activity and neuromuscular maturation in the offspring, which was related to delay in body weight gain. However, the development of reflexes and visual discrimination was not affected by maternal exposure to 3,3',4,4',5-pentachlorobiphenyl (Bernhoft et al. 1994). Taken together, these data may indicate that highly toxic coplanar PCBs may not be direct developmental neurotoxins in rodents. If this is the case, it is questionable if the use of individual PCB congeners in studies on the effects on neurochemical and behavioral development will resolve novel structure-activity relationships within the same time-frame as the structureactivity relationships for immunotoxicity or CYP1A1 induction in adult animals. First, the reproductive studies involved are much more lengthy and costly than acute studies with adult animals. Secondly, there is no general agreement on experimental protocols (timing and length of PCB administration, neurochemical and behavioral endpoints) between researchers working in this field, frustrating the comparison of data. Thirdly, it is very likely that complex interactions of Ah-receptor binding, PCB metabolism, fetal accumuation of metabolites and hormonal alterations affect brain development in vivo, so that the structure-activity relationships for individual congeners will not predict the effects of complex mixtures.

Therefore it may be more useful to characterize the effects of environmentally relevant mixtures in terms of dose-response studies, neurochemical and behavioral endpoints and species sensitivity. Environmentally relevant mixtures can be obtained by extracting contaminated foodstuffs or constructing mixtures using synthetic standards. Although mixtures will vary somewhat in their composition, the results of such studies may be more relevant for regulatory purposes than data based on studies with individual congeners. Cell culture techniques using glial cell or dissociated neural cell cultures may be useful in investigating structure-activity relationships of the direct effects of individual PCB congeners on brain development. Parameters that have been first demonstrated to be affected in vivo should be analysed in vitro.

Main conclusions:

1) PCB congeners (3,3',4,4'-tetrachlorobiphenyl, 2,3,3',4,4'-pentachlorobiphenyl and 2,3',4',4,5-pentachlorobiphenyl) can be metabolized to hydroxylated metabolites which accumulate in the fetal plasma and brain and cause severe reductions in late gestational fetal plasma and brain thyroxine levels in rats.

2) The reductions in brain T 4 levels in late gestational fetal rats are effectively compensated by increases in Type II thyroxine 5'-deiodinase activity, so that only marginal decreases in brain T 3 levels are observed following maternal exposure to a commercial PCB mixture. This is an indication that PCB-induced decreases in plasma T 4 levels are not responsible for alterations in the development of the central nervous system.

3) maternal exposure to the commercial PCB mixture (Aroclor 1254) specifically alters the development of serotonin metabolism in the brain of the offspring in rats. Since the dopamine metabolism exhibits a greater sensitivity and persistency for the administration of Aroclor 1254 in adult rodents and macaques than serotonin metabolism, the mechanism of PCB-induced developmental neurotoxicity is distinct from the mechanism of alterations in biogenic amine metabolism in adult animals.

4) The development of both neuronal and glial cells is affected in the brains of offspring from pregnant rats treated with Aroclor 1254. The alteration in astrocyte development in the brainstem of PCB-exposed offspring is not a response to neuronal death, for levels of glial fibrillary acidic protein (GFAP) are decreased, while increased neuronal death is generally accompanied by increases in GFAP expression. It is therefore likely that PCBs affect brain development by altering cell differentiation and proliferation.

5) Since the brainstem is one of the first structures to develop in the brain, the observed alterations in brainstem development following pre- and postnatal PCB exposure probably have a negative effect on the subsequent development of other brain structures.

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