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Work-related respiratory health complaints were reported in a potato processing plant. Responsible agents and the underlying mechanism for these disorders were unclear. The first aim of this study was to quantify the occurrence of work-related respiratory health effects in workers of all four plants of the potato processing company. The second aim was to study three possible mechanisms: a Type I allergy to potato, a Type III allergy to microbial components or potato (Hypersensitivity Pneumonitis), and a non-specific airway inflammation caused by microbial components such as bacterial endotoxin.Sixteen percent of the 142 workers studied indicated work-related respiratory symptoms on a questionnaire. Specific IgE to airborne dust extract could not be detected in workers' sera, and therefore a Type I allergy was highly unlikely to play a role in the etiology of work-related respiratory effects. Specific IgG to airborne dust extract was detected in sera of nearly all workers. This humoral immune response was dominated by the IgG4 subclass, the latter being present in about half of the workers. The antigens involved appeared to be heat-labile potato proteins. No association was found between specific IgG and the prevalence of work-related respiratory symptoms.On the basis of these findings it was judged improbable that a Type III allergy and with that Hypersensitivity Pneumonitis played a predominant role in the etiology of work-related respiratory effects. High levels of airborne bacteria and endotoxin were found, being significantly related to acute lung function changes. This was observed both for spirometric lung function change across the Afternoon shift, and for peak flow changes across all three types of work shifts. Chronic respiratory effects were not observed in cross-sectional analyses, which might be partially due to a health-related selection in this industry.Results in this thesis strongly suggest a predominant role of non-specific airway inflammation caused by bacterial endotoxin in the etiology of acute respiratory effects. Recycling of process water contributes to bacterial growth and hence to the build-up of endotoxin. Control measures to reduce endotoxin exposure should be evaluated, with a priority for elimination and/or reduction at the source.

SummaryRenal proximal tubular (RPT) cells can be exposed apically to glomerulary filtrated and basolaterally to non-filtrated nephrotoxic compounds. To excrete these compounds via the urine, RPT cells are equipped with transport systems able to transport nephrotoxicants from the basolateral to the apical side of the tubule. Since the majority of in vitro models used so far to study nephrotoxicity concern suspensions of RPT cells or RPT cells cultured on solid supports, in which transport systems are only partially or no longer active, less attention has been paid to the role of these transport systems in renal toxicity. An adequate assessment of RPT toxicity of nephrotoxicants should include the role of functional transport systems. Therefore, the aim of the present thesis was to develop such an in vitro model. We cultured rat RPT cells to confluency on porous supports of tissue culture inserts. Monolayers of RPT cells remained confluent for 24h. After that time, they became leaky and cell viability gradually declined (Chapter 2).The glutathione (GSH) conjugates of (hydro)quinones, nitrobenzene and ethacrynic acid were used as model compounds. In vivo, these compounds are targetted to RPT cells since these cells are equipped with both enzyme and transport systems that use these potentially toxic glutathione conjugates as substrate.The glutathione conjugate of the quinoid compound menadione (MGNQ) was only toxic after basolateral challenge of confluent monolayers of RPT cells (Chapter 2). By preventing basolateral uptake of this compound via organic anion systems, an increase in cytotoxicity was observed. Similar effects were observed when basolateral uptake and subsequent deacetylation of M(NAC)NQ, the N -acetylcysteine conjugate of menadione, were inhibited. It is generally agreed upon thatγglutamyltranspeptidase (γGT) and dipeptidase mediated metabolism of GSH conjugates into cysteine conjugates is a prerequisite for cellular uptake of the latter conjugates. MGNQ induced apical cytotoxicity only after inhibition of apicalγGT. These observations suggest that cellular uptake of thioether conjugates is associated with detoxification. The postulated reaction mechanism resulting in detoxification of the GSH and N -acetyleysteine conjugate of menadione is drawn in Fig. 4 (Chapter 2).Detoxification of quinone cystein(glycinyl) conjugates can occur via an intramolecular cyclization reaction which eliminates the reactive quinone function (Monks et al. , 1990). The identification and characterisation of the cyclization product of MGNQ provided evidence that this reaction is responsible for the detoxification of MGNQ by RPT cells in this in vitro system (Chapter 4).In agreement with results obtained with MGNQ, both mono-substituted glutathione conjugates of 2- tert -butyl hydroquinone (5SG-TBHQ and 6SG-TBHQ) were only toxic after basolateral exposure of RPT cell monolayers (Chapter 3). Apparently, at this side of the monolayerγGT-mediated metabolism of GSH conjugates is probably not sufficient for complete detoxification of these conjugates in this in vitro system (Chapters 2 and 3). In addition, the di-substituted GSH conjugate of TBHQ (3,6SG-TBHQ) was not cytotoxic in our system. In line with this, 3,6SG-TBHQ has a lower ability to generate reactive oxygen species compared with both monosubstituted conjugates (Van Ommen et al. , 1992), suggesting that the (hydro)quinone induced proximal tubular toxicity mainly results from redox cycling (Chapter 3). Since superoxide dismutase and catalase, which cannot be transported into the cell, completely abolished SG- TBHQ induced cytotoxicity, quinol-thioether induced redox cycling is likely to take place extracellularly (Chapters 2 and 3).When RPT cells were cultured on solid supports, apical exposure to 5SG-TBHQ induced a cytotoxic effect whereas 6SG-TBHQ did not. Inhibition ofγGT completely abolished 5SG-TBHQ induced cytotoxicity, whereas a cytotoxic effect was observed with 6SG-TBHQ. This result underlines the concept thatγGT on the one hand initiates detoxification of GSH-conjugated (hydro)quinones by removing the reactive quinone function (5SG-TBHQ) and on the other hand it gives rise to conjugates that can undergo redox cycling more easily (6SG-TBHQ) (Chapter 3).No transepithelial transport of MGNQ or metabolites thereof could be observed across RPT cell monolayer. Most likely, transport occurred via paracellular leakage as a result of a reduction in monolayer integrity due to toxicity via extensive redox cycling of the quinone under the culture conditions. Also, no basolateral to apical transport of the GSH conjugate of ethacrynic. acid, anα,βunsaturated ketone (EASG), was observed across the RPT cell monolayer. Probably, the transporter responsible for transport of this conjugate lost its function in our model. In contrast, only apical to basolateral transport of EASG was observed. Since acivicin reduced this transport of EASG and inhibited the formation of free, unconjugated ethacrynic acid (EA), not the GSH conjugate is transported but rather the cysteine conjugate and/or free EA (Chapter 4).In Chapter 5 it was observed by means of 19F-NMR that monolayers of both LLCPK 1 cells and primary RPT cells were not able to metabolise the GSH conjugate of 2,5-difluoronitrobenzene (FGNB) into its N -acetylcysteine derivative (FNAcNB). Consequently, the cysteine conjugate of 2,5- difluoronitrobenzene (FCysNB) was the major metabolite formed. However, freshly isolated RPT cells were able to form mercapturic acids but, upon cultivation, quickly lose this capacity. In addition, no apical to basolateral transport of the cysteine conjugate of 2,5-difluoronitrobenzene (FCysNB) was observed across monolayers of primary RPT cells (Chapter 5).In Chapter 6 we studied the ability of another in vitro renal cell system i.e. freshly isolated renal cortical slices to metabolise xenobiotics by measuring their N -acetylation capacity. We observed that these slices metabolised F Cys NB into the mercapturate F N AcNB. To find out whether cryopreserved rat renal cortical slices could be used as an alternative for freshly isolated tissue in metabolism studies, we compared their biotransformation capacity. For up to 4 h, the N -acetylation capacity of both type of slices is comparable. Thereafter, the N -acetylation capacity is substantially lower in cryopreserved tissue. Since cryopreserved rat renal cortical slices did not metabolise F G NB into a lower amount of F Cys NB than freshly isolated tissue, cryopreservation did not affect the activity ofγGT and dipeptidases.Since uptake of xenobiotics via organic anion transporters is another determinant of biotransformation capacity of RPT cells, we studied organic anion uptake of both type of slices using para-aminohippuric (PAH) acid as model compound. Compared with freshly isolated slices, cryopreserved rat renal cortical slices exhibited similar PAH uptake characteristics.Concluding remarksSelective exposure of confluent monolyers of rat renal proximal tubular cells to glutathione conjugates of the tested quinone and hydroquinones revealed that these compounds were more toxic after basolateral exposure than after apical exposure. In addition, the observed cytotoxicity appeared to be the result of mainly extracellular redox cycling of tested (hydro)quinones. The physiological significance of the observed effects remains obscure since our in vitro model suffers from several artefacts.For instance, we could not observe basolateral uptake of GSH conjugated menadione and ethacrynic acid. In addition, the results obtained with GSH conjugated 2,5-difluoronitrobenzene (FGNB) suggest that apical uptake of cysteine- S -conjugates is also absent in our RPT cell monolayer. Probably as a result of the fact that the latter characteristic is a prerequisite for N -acetylation of these cysteine-S-conjugates by RPT cells, we were not able to measure N -acetylation of these conjugates. As a consequence of non- functional cysteine transport, large amounts of (hydro)quinone-thioethers remain outside the RPT cell. Since hydroquinones readily autoxidize under culture conditions, cytotoxicity emerges as a result of extracellular redox cycling.In contrast to cultured RPT cells, the N -acetylation capacity was present in freshly isolated RPT cells. In another in vitro model it was shown that freshly isolated rat renal cortical slices also metabolised FGNB into its N -acetylcysteine conjugate. However, cryopreservation of rat renal cortical slices affected its N -acetylation capacity. Similar effects of cryopreservation were observed for organic anion transport by renal cortical slices.In summary, RPT cell monolayers are useful for studyingγGT and dipeptidase mediated metabolism of glutathione conjugates. With respect to metabolic capacity, freshly isolated renal cortical slices provide a more appropriate tool for investigating xenobiotic metabolism in vitro. However, the results obtained from both in vitro models underline the necessity to characterise renal biotransformation and transport systems involved in renal metabolism of xenobiotics. In addition, functional limitations of the in vitro model used have to be discerned. In order to improve the usability of RPT cell cultures it is of paramount importance to evaluate the influence of culture environment on the expression of differentiated cellular characteristics.

The principal lesion characteristic of atherosclerosis is the plaque. This lesion mainly consists of smooth muscle cells, connective matrix and large amounts of extracellular lipids. Smooth muscle cell hyperplasia is an integral event in atherosclerotic plaque formation and the resultant occlusion of blood vessels. These abnormal cell proliferations are primarly caused by defects in the autocrine and/or paracrine growth regulation. A novel mechanism critical to atherogenesis introduced in the present study is gap junctional mediated growth control. The atherogen-induced effects on gap junctional communication between human smooth muscle cells are described in chapters 2,3 and 4. The role of autocrine growth stimulation in atherogenesis is shown by the demonstration of the presence of activated transforming genes in atherosclerotic lesions (chapter 5) and the cell transforming potential of (atherogenic) low density lipoproteins (LDL) as shown in chapter 6. Chapter 5 also demonstrated that several transforming genes may be present in human plaque DNA, one of them being PDGF-A (platelet-derived growth factor chain A). The expression of the latter gene can be modulated by oxidized LDL (chapter 7).The present investigation provides additional evidence for the hypothesis that autocrine production of growth modulating factors may contribute to the characteristic abnormal smooth muscle growth in atherogenesis. We demonstrated that lipoproteins with atherogenic potential, such as LDL, in contrast to the non-atherogenic HDL, are able to transform fibroblasts in vitro. Genetical alteration of intimal cells present in atherosclerotic lesions was demonstrated using DNA-mediated transfection techniques. It could be demonstrated that human aortic plaque DNA contains active transforming genes, as PDGF-A. This is in agreement with studies by Penn et al. (1986) and Ahmed et al. (1990), showing transforming genes in human coronary artery plaques. Even DNA of cultured human atherosclerotic plaque cells appeared to have cell transforming capacities (Parkes et al., 1991). Only Yew et al. (1989) could not demonstrate a transforming potential of human plaque DNA. The variable results obtained sofar might indicate that different mechanisms are involved. It should be stressed in this respect that in all these studies pooled plaque samples were used and it should be realized that the transforming potential is not necessarily present in each plaque. The observed transforming potential of plaque DNA supports very well the monoclonal hypothesis of Benditt and Benditt (1973). They observed that atherosclerotic lesions start as singular focal masses containing monoclonality and suggested that these masses were in general similar to benign tumors as observed in other tissues. However, it has been argued that apparent monoclonality could also arise because of strong selection pressure favoring a subpopulation of cells, although findings of transforming genes, such as PDGF-A, present in atherosclerotic lesions and the transforming potential of atherogens indicate the opposite. Additional arguments in favor of the monoclonal hypothesis are that tumor initiators like chemical mutagens and promutagens (Albert et al., 1975; Bond et al., 1981), radiation (Gold, 1961) and oncogenic viruses (Minick et al., 1979) can induce atherosclerotic lesions in laboratory animals. Hence, molecular alterations underlying the proliferation of smooth muscle cells could show resemblance to the molecular events, which are critical in the development of cancer.The current view on atherosclerosis is based on the "response to injury" hypothesis, implying a paracrine stimulation of smooth muscle growth as a result of injury of endothelium (Ross, et al., 1976; Ross, 1981). Most of the studies of experimental atherogenesis are implemented in the framework with this hypothetical model. Support for this hypothesis is based on the following observations. Atherosclerotic lesions can be induced in experimental animals by endothelial denudation and factors derived from platelets can induce smooth muscle growth in vitro. Injury to arterial endothelium by mechanical, chemical, toxic, viral, or immunological agents caused endothelial denudation, and was followed by platelet adhesion and aggregation, with consequent release of platelet-derived growth factors (PDGF), in turn leading to migration into and proliferation of smooth muscle cell in the intima and secretion of connective tissue components. Over the years, the hypothesis has been modified (Ross, 1990). First of all, it became apparent that actual denudation was not a consistent early feature of atherosclerosis (Davies et al., 1976). Secondly, platelet adherence is neither necessary nor sufficient to cause the lesions (Schwartz and Reidy, 1987). Presently, the view is: a. the endothelium can respond to a variety of stimuli, by subtle changes in function (endothelial dysfunction) and/or by the induction of new endothelium properties; thus non- denuding injury may be important in initiating the lesions of atherosclerosis and b. platelets are not the sole initiators of smooth muscle proliferation, since growth promoting and growth inhibitory factors secreted by other cell types, including macrophages and endothelial cells, may modulate smooth muscle growth.However, not all observations made can be explained by the "response to injury" hypothesis, including the findings that (intimal) smooth muscle cells themselves can secrete and can respond to growth modulating factors and the cell transforming potential of plaque DNA as demonstrated above, thus implying an involvement of autocrine growth stimuli. A good candidate for autocrine growth factor stimulation of smooth muscle cells is PDGF-A. High transcript levels of this transforming gene were detected in intimal cells of human atherosclerotic lesions (Libby et al., 1988; Wilcox et al., 1988). The present study showed that PDGF-A was identified as one of the transforming genes present in human plaque DNA and the expression of this gene can be induced by LDL (oxidized). HDL, a protective lipoprotein to atherosclerosis, did not modulate PDGF-A transcript levels in smooth muscle cells. While both oxidized LDL as well as PDGF-A transcripts are detected in atherosclerotic lesions this molecular mechanism could play a role in atherogenesis.Furthermore, the present study provides strong evidence that disturbance of intercellular communication is another mechanism, which is probably important in the etiology of atherosclerosis. It is shown that compounds with atherogenic potential inhibit communication between human smooth muscle cells. The potency to modulate cell-cell communication correlates well with their atherogenic potential. For example, low density lipoproteins (LDL) in oxidized form and cholesterol oxidation products inhibited cell-cell communication. A non-atherogenic lipoprotein as high density lipoprotein (HDL) did not influence gap junctional communication between human smooth muscle cells. Otherwise, growth factors like PDGF and EGF can modulate cell-to-cell communication (Maldonado et al., 1988; Madhukar et al., 1989). This would imply that both paracrine and autocrine growth factor production are involved in atherogenesis, in which their influence on the gap junctional mediated growth control mechanism is a vital step in atherogenesis. At this stage, disruption of intercellular communication results in an uninhibited multiplication of these cells leading to the formation of (monoclonal) atherosclerotic lesions.The main conclusion of the present study is that the three phenomena 1) production of growth modulating factors by vascular cells, including endothelium, monocytes, platelets and smooth muscle cells 2) DNA modification (e.g. PDGF-A) of smooth muscle cells 3) loss of intercellular communication together are important factors in the process of atherosclerosis. The two main theories on atherosclerosis, the "monoclonal" and the "response to injury" theory, are compatible in many respects and can be fit into one unifying hypothesis as described in Fig. 8.1The figure shows that one of the earliest events in atherosclerosis is an increased adhesion of monocytes to what appears to be intact arterial endothelium, a phenomenon which can be well demonstrated using scanning electron microscopy (Faggiotto et al., 1984). In this process lipid factors like hypercholesteraemia and other factors as IL-1 or TNF (which introduces adhesion molecules. on endothelial cells) are important triggers. As a consequence of monocyte emigration, there is focally increased permeability to LDL and macromolecules (Territo et al., 1984; Gerrity et al., 1979). An altered endothelial function (dysfunction) causing increased permeability may also be induced by risk factors such as hypertension, hyperlipidaemia, smoking, immunological factors, stress and diabetes mellitus (Reidy, 1985; Sieffert et al., 1981; Gordon et al., 1981, Munro and Cotran, 1988). In this way the agents present in the bloodstream (such as LDL) accumulate and are modified in the arterial wall and thereafter exert their effects locally on vascular cells.The next important step could be DNA modification(s) of smooth muscle cells as shown by the presence of transforming genes, as PDGF-A, in lesion DNA and the effects of the atherogenic LDL on cell transformation. This defect of an autocrine growth regulation is followed by another vital growth control mechanism, the gap junctional communication. Communication between human smooth muscle cells has been modified by several atherogens, like LDL (oxidized) and oxysterols. In case of impairement of both growth regulation mechanisms clonal growth may occur.The processes described on the left side of the scheme are consistent with the paracrine proliferation mechanism of "the response to injury" theory of Ross (1990). The first trigger in this process is a dysfunction of the endothelium. This process introduces the adhesion and aggregation of platelets and/or monocytes to the endothelium, which results in a release of growth modulating factors as platelet-derived growth factor. At this stage, paracrine growth stimulating factors do not yet cause abnormal smooth muscle growth, because autonomous growth of cells still is controlled by gap junctional communication with surrounding cells. Only when growth factors (and atherogens) inhibit gap junctional communication abnormal smooth muscle cell proliferation occurs. Thus, inhibition of gap junctional intercellular communication could play a predominant role in the onset of atherogenesis.A better understanding of these growth modulating processess will improve the possibility to study the possible atherogenic properties of chemicals within the framework of toxicological research.

The main objective of the present study was to evaluate the possible impact of pesticides on epigeal arthropods in arable land. It was also envisaged to develop a predictive model for possible undesirable effects of pesticides on the epigeal arthropod fauna using an indicator species from the field. The strategy was the following. In the field, species were identified that were (1) sensitive to a number of pesticides, (2) abundant, (3) regular in time and space and (4) easy to sample and identify (Phase I). Two species, the erigonid spiders Oedothorax apicatus (Blackwall) and Erigone atra (Blackwall) were selected for toxicity studies in the laboratory using deltamethrin as a model compound (Phase II). The results of these studies were used to develop a predictive model which was tested under field conditions (Phase III).Phase I: Initial Field ObservationsPhase I was implemented in the South Flevoland Polder, in cooperation with the State Authority for the Development of the Lake IJssel Polders. Observations on the above-ground arthropods were conducted in 17 commercially farmed fields and in one experimental field, from March 1985 to November 1986, during two growing seasons and one winter. The arthropods were sampled by pitfall traps in oilseed rape fields which were treated with a number of insecticides and herbicides, and in cereal fields which were either treated with herbicides or weeded chemically against herbs, at recommended dosages. In the experimental field, dosages were varied and the effect of ploughing was observed. During the treatments in situ bioassays were carried out with captive spiders. From @ 5100 pitfall samples over 200,000 specimens were identified, belonging to 147 taxa. The observed short term effects were the following. A limited number of species were significantly affected by the treatments with insecticides. These were the carabids Clivina fossor after deltamethrin applications and Trechus quadristriatus after fenitrothion, and 7 spider species. The latter were reduced after treatments with either deltamethrin, fenitrothion or bromophos-ethyl. No effects were observed of herbicide treatments. It was also demonstrated, however, that the effect of mechanical disturbance by ploughing was comparable to the strongest effects of insecticides. It was concluded that an assemblage of spider species consisting of Oedothorax apicatus , Erigone atra , E. dentipalpis , Meioneta rurestris and Bathyphantes gracilis may be a sensitive indicator for early detection of side-effects of insecticides. This group represents 80-90% of the erigonid and linyphiid fauna of arable land and they are common throughout North-Western Europe. The most abundant species, E. atra and O.apicatus were selected for further study.Possible long-term effects on the spider fauna have been studied in a transversal study in fields with different cultivation histories. The differences were related to the distance between fields rather than to cultural history. There was one exception: in cereal fields that have, since reclamation, been weeded by harrowing, the spider fauna was considerably poorer than in herbicide treated fields.The effect of deltamethrin on the spiders was observed several times, under different weather conditions. It appeared that the toxicity was highest under hot and dry conditions and low under cold and wet circumstances. This was not in agreement with the known negative temperature-toxicity relationship of pyrethroids and the higher availablity of chemicals from wet surfaces. This relation, therefore, was studied in more detail in the following phase.Phase II: Toxicity studiesThe first step in the analytical phase was the development of new methods for rearing of and toxicity testing with the selected indicators. 0. apicatus females appeared to be the most appropriate species both for rearing and testing.The second step concerned the availablity of deltamethrin to this species under varying environmental conditions. Three routes of exposure were examined: topical application (simulating exposure to spray); uptake via food, and exposure to contaminated surfaces (soil or glass filter). Part of the work was carried out with radio-labelled deltamethrin. It appeared that uptake of residues from a surface by the tarsi is the most important route. Field observations carried out with a fluorescent tracer showed that the direct exposure of the spiders and their prey species to the spray, was very limited. This indicated that in the field as well, direct exposure and oral uptake are of limited importance. The availability of the residue, however, strongly depends on the water content of the soil. A decrease of the soil moisture content to 66 % of the field capacity resulted in a drop of 75 % in availability.The third step concerned the relation between environmental factors, i.e. climate and animal antagonists, and the toxicity of deltamethrin. Toxicity tests carried out under varying moisture and temperature conditions showed that at decreasing air humidity the toxicity increased, at all temperatures tested. The compound is most toxic at a combination of a low air humidity or the absence of drinking water and a high temperature. A negative temperature-effect relationship in the range 10 - 30 °C could be observed only at maximum air humidity. The behaviour of the spiders with regard to the moisture in the environment changed after exposure to sublethal dosages of deltamethrin. Spiders not visibly affected showed an increased preference for wet conditions. Affected spiders, on the other hand, showed a reduced capacity to select moist conditions. Their capacity to escape from predators was also reduced. Analysis of gut contents of the main predator, the carabid beetle P. cupreus , from the fields sampled in Phase I, showed an 8-fold increase in the consumption of linyphiids and erigonids after deltamethrin spraying. A few tests were carried out with both captive and reared spiders. There appeared to be a marked difference in sensitivity between these groups. This was primarily imputed to differences in selection mechanisms between laboratory and field.With these results a regression equation was composed which predicts the toxicity of deltamethrin to O. apicatus females under various weather and soil moisture conditions.Phase III. Validation under field conditionsSimilarly to Phase 1, the field observations of Phase III were carried out in the South Flevoland Polder. A 75 ha field was divided into 27 test plots. These plots were treated three times at different dates, in three replicates, with three dosages of deltamethrin. The following observations were carried out: acute effects in in situ bio assays, activity-density of E. atra and O. apicatus ; deposition of deltamethrin on the soil, on the spiders, on carabid, and on collembola (the main food items of the spiders). Meteorological data were obtained from a near-by meteo station and moisture of the top soil was calculated from data on rainfall, evaporation, and characteristics of the soil and the crop. Only E. atra males were abundant enough at the first and the last spraying date to allow calculation of dose-response relationships. The angles of the regressions appeared to differ for both dates. However, comparison of the observed reduction in O.apicatus females and E. atra males with the toxic effect predicted for O. apicatus females, showed that the laboratory based model had a fair predictive value for the given situation. Apparently erigonid spiders are good indicator species for the assessment of possible undesirable effects of insecticides.

Tuberiferous and nontuberiferous wild Solanum species are increasingly being used in potato breeding as a source of genes for disease and pest resistances and for other valuable characteristics. A disadvantage of Solanum species, from a consumers point of view, is that they contain steroidal glycoalkaloids (SGAs), which are natural toxins occurring in all parts of these plants. The SGAs consist of a C 27 -steroidal alkaloid (SA) and a sugar moiety, often a tri- or tetrasaccharide. The tubers of the cultivated potato usually contain small quantities of one type of SGAs, the solanidine glycosides.Evidence has been presented that utilization of wild Solanum species can result in the introduction of hazardous levels of solanidine glycosides into the cultivated potato. However, little is known of the qualitative and quantitative SGA composition of wild Solanum species used in potato breeding. Such information on hybrid offspring or on cultivars containing germplasm from wild species is entirely lacking.The aim of the studies described in this thesis was to evaluate the possible health hazards of the SGAs from the genus Solanum and to develop and apply methods for analysis of the SGA compositions of Solanum species. The collected information was placed into the perspectives of potato breeding and of food safety, in order to point out possible consequences of introducing undesired levels or types of SGAs into the cultivated potato.In Chapter I, the literature on the distribution and accumulation of solanidine glycosides in the cultivated potato and on the biosynthesis of the SGAs is reviewed. It was shown that many factors during the growth and post-harvest period of potatoes can lead to levels of solanidine glycosides exceeding 200 mg/kg fresh weight. This information is important also with respect to introduction of SGAs from wild Solanum species into the household potato, as alien SGAs will almost certainly accumulate similarly, because the biosynthetic pathways of the various SGAs are closely related.The objective of Chapter II was to evaluate the toxicity of the SGAs, based on the data available in the literature, in order to assess possible consumer hazards and to derive safe levels for household potatoes. The data showed that acute poisoning in man may occur due to consumption of potatoes with a solanidine glycoside content above 200 mg/kg fresh weight. This level is only two to three times higher than levels that are considered normal for tubers of current cultivars. Because of the virtual absence of chronic toxicity data, an adequate 'no-adverse effect level' for potato SGAs could not be assessed. Consequently an acceptable daily intake (ADI) figure for man or an acceptable level for potatoes could not be derived. It was concluded that the SGA content of new household potato cultivars should not be allowed to rise above the average level of the current cultivars; preferably it should be lower. SGAs alien to S. tuberosum must not be introduced into the household potato as acceptable levels have not been established.The methods for SGA analysis described in the literature are not adequate for determining -qualitatively and quantitatively- the SGA composition of breeding material. Therefore, a comprehensive, quantitative and efficient method applicable to diverse plant material, was developed.In Chap ter III, a new hydrolysis technique employing a two-phase system is described. The technique was developed in order to prevent the losses of aglycones which do usually occur during conventional hydrolysis of different SGAs. The two-phase system consists of an aqueous acid phase, in which the glycosides are hydrolysed, and an immiscible nonpolar organic phase, which serves as a protective phase for the unstable nonpolar aglycones. Using the technique quantitative recoveries of various SAs after simultaneous hydrolysis of different SGAs were obtained.In Chapter IV a capillary gas chromatography (GC) method is described, which enabled -for the first time- the separation of the Δ5-and 5α-aglycone pair solanidine and demissidine as well as separation of the other SAs studied. Derivatization of the SAs was not required. The capillary GC method in combination with the bisolvent extraction and two- phase hydrolysis described in Chapter III offered prospects for quantitative analysis of complex SGA compositions of Solanum species in a single run.Chapter V deals with the development of a method that enabled an unambiguous differentiation during capillary GC between peaks of unknown SAs and peaks of closely related nonnitrogen-containing (non-N) compounds such as sterols and steroidal sapogenins. The method involves a splitting device that connects a capillary column to a dual detector system, for flame ionization detection (FID) and N-specific detection (NPD), respectively. This system is connected to a two-channel system for interactive processing of the two sets of data. The application of hydrogen as carrier gas was introduced to achieve a short analysis duration. As tubers of wild Solanum species appeared to vary strongly in SGA contents, identification by retention times was not possible. Therefore a retention index system for the SAs was introduced. The retention indices were sufficiently reproducible, even for identification of the closely eluting Δ5-and 5α-aglycone pair solanidine and demissidine. Application of NPD/FID response ratios together with retention indices proved to be useful in the identification and characterization of SAs. Costly spectrometric techniques will have to be employed for characterization only when new SAs are detected.Chapter VI deals with an evaluation of the developed methods using diverse plant tissues of wild Solanum and Lycopersicon species, cultivars and hybrid progeny. The results showed that the methods were useful for qualitative and quantitative analysis of the SAs in the diverse samples. A potato extract spiked with 20 SAs, including three Δ5-and 5α-aglycone pairs, could be analysed in a single GC run. A comparison with procedures described in the literature showed that the procedure developed for sample preparation followed by capillary GC should be applied, because literature procedures for sample preparation can lead to losses of various SGAs or SAs, and other chromatographic techniques do not offer the efficiency required for separation of the various SAs.In Chapter VII, the SGA compositions of a large number of wild Solanum species used in potato breeding, are presented. The tubers of most of the species contained high concentrations of solanidine glycosides. Some species contained glycosidic-bound demissidine, solasodine and/or tomatidine. Unidentified compounds were also detected, which were most probably SAs as was shown by their NPD/FID response ratios. The total SGA contents of the tubers of the wild species varied from 123 to 7348 mg/kg. In order to place these contents in a realistic perspective, tubers of cultivars, corresponding in small size with and grown under the same conditions as the tubers of the wild species, were analysed. The contents of solanidine glycosides of these tubers were two to three times higher than those of field-grown normal tubers. Thus, even when this factor was taken into consideration, the SGA contents of the tubers of several wild species were extremely high. This study also revealed that samples of some wild species contained high or low levels of one or many other glycosidic-bound SAs, and that the SGA composition can vary within species and between different organs of one plant. The results further suggested that the SGA synthesis can be organ specific or regulated by light and that translocation of SGAs from leaves to tubers and viceversa did not occur. It was concluded that wild crossing parents should be analysed as to their SA composition, before they are used in a breeding programme.A procedure developed for the characterization of unidentified SAs present in plant extracts with complex SA compositions, is described in Chapter VIII. The combined results of GC-mass spectrometry (MS) applying electron impact and chemical ionization, and high-resolution MS, of the application of retention indices and NPD/FID response ratios, and of a comparison of the degree of dehydration of SAs in different twophase hydrolysis systems, were used. This study revealed that the SAs all possessed a solanidane skeleton; they were characterized as substituted, e.g. hydroxylated or methylated, forms; dehydrogenated forms; or substituted saturated forms of solanidine. Most of these SAs had not been reported before. The complete SA composition, including the minor new SAs, of Solanum (sub)species used in potato breeding, is presented in this chapter and the importance of analysis of the minor SAs is indicated.S. vernei is widely being used in potato breeding. Although the SGA composition of its tubers had not been reported, cultivars containing S. vernei germplasm have been released and an increasing number will be released in the future.Chapter IX describes the identification and characterization of the SAs of S. vernei tubers produced under various growth conditions, and deals with a tentative study on the transmission of these SAs to hybrid offspring. High levels of solanidine and solasodine glycosides were found together with tomatidenol and novel glycosidic-bound SAs, amongst which the 22R,25R epimer of solanidine, a structural configuration not reported before for naturally occurring solanidanes. It was revealed that the SA composition can vary significantly in tubers grown under different cultivation conditions.In the tubers of S. vernei offspring, high levels of solanidine glycosides were found but the newly identified and characterized SAs of S. vernei were not detected. Solasodine was also found in the offspring, even in tubers of cultivars obtained after several times backcrossing, but fortunately, the levels were low. However, it can not be totally excluded that S. vernei offspring may synthesize hazardous levels of solasodine glycosides under particular growth or post-harvest conditions, and in this respect the possible teratogenic potency of solasodine should be kept in mind.In conclusion it can be stated that the ingestion by the public of the amount of solanidine glycosides should not be allowed to rise but should preferably be reduced. SGAs alien to S. tuberosum should not be introduced into the household potato. The basis for the production of a potato crop safe for consumption is, to grow current cultivars and to breed new ones which accumulate low levels of only solanidine glycosides under various growth and post-harvest conditions. Utilization of germplasm from wild Solanum species must be approached with caution. Therefore it is recommended to analyse the SGAs of potential wild crossing parents before they are used in a breeding programme, in order to select the genotypes that combine a desired trait with the least unfavourable SCA composition. Depending on the SGA composition of the parents, the offspring should be monitored too.For analysis of SGA compositions, the newly developed procedures for separation, quantification and identification/characterization, described in this thesis, must be applied, as the conventional methods described in the literature do not meet the required standards.The accumulation of SGAs in new cultivars containing wild-species germplasm should be studied under various environmental conditions before such cultivars are registered. The guideline of 60-70 mg solanidine glycosides per kg fresh tuber recommended in the literature from the viewpoint of consumer safety (see Chapter II), should be used in potato breeding until an adequate acceptable level for these compounds has been established. Van nature komen in alle delen van planten van het geslacht Solanum steroid-alkaloidglycosiden (SGAs) voor. Het beschreven onderzoek had tot doel de potentiele gezondheidsrisico's van de SGAs te schatten en methoden te ontwikkelen, om de SGA-samenstelling van Solanumsoorten te kunnen analyseren. De verzamelde informatie werd geevalueerd, vanuit het oogpunt van zowel de aardappelveredeling als van de voedselveiligheid, teneinde de mogelijke gevolgen aan te geven van het introduceren van ongewenste gehalten of typen SGAs in de gecultiveerde aardappel

This thesis is concerned with exposure of stone loach ( Noemacheilus barbatulus L.), a fairly common fish in British rivers, to cadmium and lead. The work described in this thesis was performed to assess the relative importance of different routes of entry (water, food and sediment) on the body burden of cadmium and lead in stone loach under field conditions. These studies were based on the premise that metabolic rate of stone loach is an important factor affecting metal burden. Metabolic rate in fish is dependant on factors which include body weight, temperature, and physiological condition. Therefore, a wide weight range of fish was deliberately used in this study, to obtain information on the effect of body weight, in contrast to common practice in laboratory studies where uniformity of body weight is used to reduce the variation in effect on uptake of pollutants.This thesis is divided into the following parts:- introduction (part I);- laboratory studies (part II);- field studies (part III);- mathematical modelling (part IV);Part I of this thesis describes where lead and cadmium are present in the environment and the effect of human activities on distribution of the metals. This distribution was studied in areas in Derbyshire (UK), where one site (the River Ecclesbourne with mineral veins at the head of the valley), had higher metal levels in sediment and water than two other sites (both with 'background' levels of metals). In general, uptake of cadmium and lead by fish may occur via three pathways, but there is no consensus over their relative importance, not even within one species used in different studies. The literature is reviewed on environmental fate and effects on organisms of cadmium and lead. (chapter 1).Part II (laboratory experiments) describes the laboratory experiments which were aimed at obtaining quantitative information on rates of uptake and loss of cadmium from three possible sources. Chapter 2 reports exposure to water-borne cadmium by the stone loach at different temperatures and over a range of cadmium concentrations. Rates of uptake and loss of cadmium were found to be affected by size of the fish and to increase with temperature. There was some evidence that cadmium burden of the loach reached a maximum after exposure around 16 °C. Fed fish appeared to have a higher rate of cadmium uptake from the water than had starved fish. Published rates of loss after exposure to cadmium cannot be readily compared because duration of exposure may affect rates at which a fish gets rid of cadmium.Metabolic rate was estimated by the oxygen consumption rate. Two levels were distinguished: a low 'routine' level, when the fish were at rest during light, and a higher 'active' level, when fish were swimming, which was observed for a few hours after the onset of darkness. The difference between these two levels narrowed towards the upper end of the temperature range used (6 - 18 °C), suggesting that loach became stressed at high temperatures. A similar effect of temperature on rate of cadmium uptake from Tubifex , used as food items, into fish was found. Absorption efficiency of cadmium from food by the loach decreased with increasing cadmium concentration in the food; also, in spite of increased food consumption at higher temperatures, cadmium burden of stone loach did not rise in proportion because of reduced absorption efficiency of the metal. (chapter 3).Exposure of stone loach to sediment with levels of cadmium and lead higher than 'background' increased body burdens of both metals (chapter 4). High rates of loss resulted in the rapid development of a steady state. The biological half-life for cadmium was longer than for lead in these experiments.The field studies (part III) were carried out to provide information on the growth rate of stone loach, fluctuations in the fishes' body burden and in concentrations of cadmium and lead in the environment (water, food and sediment) in relation to fluctuations in temperature with season. These field studies included a one-year sampling programme of stone loach in Derbyshire (UK) (chapter 5). The selected streams were the River Ecclesbourne, Brailsford Brook South and Suttonbrook. Growth rate of fish, which depended on temperature and age, was greatest during the spring and summer and was highest in young loach. Fish from all streams had reached their maximum length (about 110 mm) after two years and also their maximum cadmium burden. Differences in body size accounted for most of the variation in cadmium levels between loach of different age groups, but was less important for lead levels; cadmium burden fluctuated more than lead burden. Loach caught in the River Ecclesbourne had higher body burdens of both cadmium and lead; no significant differences were measured between the Brailsford Brook South and Suttonbrook.Water samples were taken from all three streams at four periods and on several occasions during each period (chapter 6). Most of the cadmium, in contrast to lead, was in solution. Flow rate of the water appeared to have a greater effect on the cadmium concentration than on the lead concentration.There was some evidence for seasonal fluctuations: metal concentrations were higher in the early spring than in the autumn.Kick-samples from the streams were examined for the range of invertebrate species. Remains of most of these species were found in the gut contents of loach, which suggests that loach are not very selective feeders on invertebrates. Cadmium levels in invertebrates showed seasonal variation: lowest levels were measured in samples taken during the spring and highest levels in those sampled during the autumn. (chapter 7).Sediment samples were taken at about 4-weekly intervals from the same sites, at the same time, as the fish. Concentrations of lead and cadmium were higher in samples from the River Ecclesbourne. Although concentrations of both metals fluctuated with sampling time, there was no consistent trend at any of the sites. (chapter 7).Part IV describes the development of a mathematical model which incorporates both laboratory and field studies (chapter 7). This model predicted adequately the range of cadmium levels found in stone loach in the field. Stone loach take up cadmium from water, food and sediment. The relative importance of these sources differed between the three sites because of differences in measured concentrations. For the R. Ecclesbourne. younger fish took up relatively more cadmium from water than did older fish; the latter received relatively more cadmium from sediment. The model predicted that consistent lower temperatures caused the metal burden to be lower during the winter period and consistent higher temperatures had the opposite effect.The results of the work presented clearly show the effect of body size on metabolic rate. The model that relates body weight (W) of fish to a dependant variable (M), e.g. cadmium burden, includes an exponent (y) as follows: yM=α* WThe values of the exponent y were comparable for respiration rate, food consumption on different rations and for cadmium burden during and after dietary exposure, during exposure to sediment and in field data. However, the value obtained from water-borne studies was lower. This suggested that, for similar values of a, water was less important as a source of cadmium than food and sediment; comparison of observed cadmium burdens during exposure in the laboratory with field data confirmed this. The values of the exponent for the lead burden obtained from field data and exposure to sediment, were lower compared to the value derived from respiration studies. This suggests that uptake and loss of lead is less affected by metabolic processes than is cadmium. Body burden of lead on exposure to sediment was comparable with that in loach from the River Ecclesbourne, suggesting that most of the lead is taken up from the sediment.Cadmium burden is higher during the winter than during the summer. This is caused by the greater effect of temperature on rate of intake than on rate of loss. The relative contribution of cadmium in water, food and sediment to cadmium burden of loach depends on environmental conditions, such as cadmium concentration in these sources, temperature and body weight. There is virtually no uptake of cadmium from water during the winter; most cadmium originates at this time from food. Particles with which most of the lead is associated and which are a prime source of lead for stone loach, are brought into suspension at higher flow rates and, therefore, exposure increased.The data in this study, compared with the published literature, suggest that the fish's handling of cadmium depends on the duration of exposure; after short-term exposure, the fish eliminate cadmium rapidly while, after longterm exposure, cadmium is not easily lost. This phenomenon may be explained by the time of exposure required for the induction of cadmiumbinding proteins. There is also some evidence that the type of exposure affects the rates of loss, presumably in parallel with the fate of cadmium within the fish. Based on a one-compartment model, the cadmium derived from food seems to remain longer in the body than cadmium originating from water and sediment. There is evidence that stone loach get rid of cadmium rapidly after short term exposure to both dietary and water-borne cadmium. This corroborates the observed fluctuations of cadmium burden in stone loach from the field.The importance of different routes of uptake of both lead and cadmium differs between fish species. Water is often regarded as the sole source of metal although there is evidence that diet can contribute significantly to the body burden of fish. Bottom feeders, in particular, seem able to acquire some metal from sediment. Moreover, concentrations in the different environmental compartments determine their relative contributions to the body burden.