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The in vivo toxicity of ozonides, possible intermediates in ozone-induced toxicity, was investigated. Methyl linoleate ozonide (MLO) (0.07 mmol/100 g body wt.), a model fatty acid ozonide, was administered to female Wistar rats either intravenously or intraperitoneally. After 24 h the rats were killed and the effects were examined. MLO was found to be toxic only after intravenous administration. The major effects were observed in the lungs. The lungs became enlarged from edema and showed severe hemorrhages. Further, total thiol was depleted in serum and lung tissue, accompanied with a significant decrease in activity of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and glutathione S-transferase. The vitamin E levels in serum and lung tissue were reduced. The malondialdehyde (MDA) concentrations in serum and lung tissue were elevated suggesting that in vivo oxidation had occurred. On intraperitoneal administration of MLO, no effects on enzyme activities, thiol and vitamin E content in lung tissue were observed. In serum, however, as on intravenous administration, an increase of the MDA levels and decreases of total thiol and vitamin E levels were found. In view of the route of administration it is to be expected that the ozonide is partly cleared by the liver, and the ozonide and its potentially toxic products are further detoxicated by vitamin E and thiols in serum before they reach the lung. The above data show that the main target organ for ozonides is the lung, and that the effects caused by MLO in vivo are in many respects similar to the effects found after acute ozone exposure. This supports the working hypothesis that ozonides may play a role in ozone-induced lung toxicity.

A further development of the coal gasification process will result in an increase of the amount of coal gasification slag (CGS). As yet little is known about the effects of storage in uncovered dumps. If there are any environmental effects, they are most likely caused by accumulation of metals from the CGS or by unacceptable physical properties of the CGS. Growth inhibition, mortality, and metal accumulation were analyzed for the plant Cyperus esculentus and the worm Eisenia fetida on CGS substrate. Pulverized fuel ash (PFA) was used as a reference. Both in the substrate and in tissues the concentrations of the cations Cu, Ni, Pb, and Zn and the anions As, B, Cr, Mo, Sb, and Se were determined. The availability of anions for C. esculentus and for E. fetida is greater in PFA than in CGS. The extent and rate of uptake of anionic metals by the plants is on the whole higher in the wetland situation. The availability of metals, expressed as the concentration factor (CF), in most cases appears to be smaller than 1 for nearly all elements. In E. fetida a CF > 1 was found only for the element As, in PFA substrate and 50% CGS. In C. esculentus a CF > 1 was found for B and Mo in the PFA substrate as well.

The biotransformation of furazolidone, both in vivo and in vitro, results in the formation of so-called bound metabolites. Following the incubation of pig hepatocytes with [14C]furazolidone, a dose- and time-related increase in the formation of bound metabolites was observed. After withdrawal of the drug from the medium, levels decreased gradually to about 50% within 36 hr. Using a newly developed method, it was shown that in the case of cells and liver microsomes at least 70% of the bound residues still contained the 3-amino-2-oxazolidinone (AOZ) side chain of furazolidone. For liver samples of piglets that had been treated orally with the drug for 10 days, followed by withdrawal periods of 2 hr or 14 days, fractions of releasable AOZ were 23 and 14%, respectively, equivalent to levels of 3.8 and 0.3 micrograms furazolidone/g of tissue, respectively. The release of the AOZ side chain of furazolidone from at least part of the bound metabolites shows their drug-related nature and highlights the requirement for both adequate withdrawal periods and the need for appropriate control methods. The relative simplicity of the newly developed method may be helpful in further establishing and controlling adequate withdrawal periods.

The cyclopentenone prostaglandin A2 (PGA2) is known to inhibit cell proliferation, and metabolism of this compound thus might be important in controlling its ultimate function. The glutathione-related metabolism of PGA2 was therefore investigated both with purified glutathione S-transferase P1-1 (GSTP1-1) and with IGR-39 human melanoma cells. Firstly, the irreversible inhibition of human GSTP1-1 and its mutants C47S, C101S, and C47S/C101S was studied. PGA2 appeared to inhibit GSTP1-1 mainly by binding to the cysteine 47 moiety of the enzyme. This binding was reversed by a molar excess of GSH, indicating that retro-Michael cleavage occurs. Secondly, after exposing IGR-39 human melanoma cells to PGA2, both diastereoisomers of the PGA2-glutathione conjugate are excreted into the medium, although with a clear excess of the S-form, due to its preferential formation by the GSTP1-1 present in the cells. Thirdly, the effect of PGA2 on intracellular GST activity was determined by quantification of the excreted glutathione conjugate S-(2,4-dinitrophenyl)glutathione (DNPSG) after exposure to 1-chloro-2,4-dinitrobenzene. DNPSG excretion was inhibited after incubation with 10 or 20 μM PGA2 for 1 or 4 hr, as a result of glutathione depletion, reversible GST inhibition, and covalent modification of intracellular GST. Furthermore, PGA2 also inhibited transport of DNPSG by the multidrug resistance-associated protein, an effect that was reversible and competitive. In conclusion, PGA2 modulates all three aspects of the glutathione-mediated biotransformation system, i.e. GSH levels, GSTP1-1 activity, and transport of GSH conjugates. A role for GSTP1-1 as a specific transport protein inside the cell is indicated. Copyright (C) 1999 Elsevier Science Inc.

Although most studies concerning the effect of vitamin A and beta-carotene on chemical carcinogenesis are focused on tumour promotion and progression, these compounds may affect initiation as well. In this study the influence of vitamin A and beta-carotene on unscheduled DNA synthesis (UDS) was investigated in hamster tracheal epithelium in organ culture exposed to benzo[a]pyrene (B[a]P). DNA-repair activities were compared with the level of B[a]P-DNA adducts as measured both by 32P-postlabeling and by immunocytochemical detection. In hamster tracheal epithelial cells, both vitamin A and beta-carotene significantly increased B[a]P-induced UDS, with 40% and 45%, respectively. At the same time, vitamin A and beta-carotene decreased the level of B[a]P-DNA adducts in these cells with 18% and 40%, respectively as measured by 32P-postlabeling and with 12% and 35%, respectively as measured by immunocytochemistry. The effect of vitamin A on B[a]P-induced UDS and DNA-adduct levels in hamster tracheal epithelium appeared to depend on the dose of B[a]P vis-à-vis the concentration of vitamin A. The results of the present study show that both vitamin A and beta-carotene cause a decrease in B[a]P-DNA adduct levels by enhancing DNA-repair activities. Because the formation of B[a]P-DNA adducts is considered to be an early step in respiratory tract carcinogenesis, it is suggested that enhancement of DNA-repair activities by vitamin A and the subsequent removal of DNA adducts may be one of the mechanisms involved in vitamin A-mediated protection against cancer.

Ever since research on the environmental impact of chemicals started in the 1950s, measures of biological responses have been considered that would now be termed “biomarkers”. Well-known examples include cholinesterase inhibition in brains of fish as indicators of possible neurotoxic effects and egg-shell thinning as an indicator of reproductive effects in birds (McCarthy and Shugart 1990; Peakall 1992). To describe these measurements, terms such as “bioindicators”, “measures of biological response”, “early warning indicators”, and “clinical measures” have been used, but have not always been defined. In this document our use of the term biomarker may include part or all of these measures; that is, the term biomarker does not refer to a new concept, but is just a new name for a monitoring principle already in existence.

Previous studies have indicated that chemicals, e.g., hydroxylated polychlorinated biphenyls (PCBs) decrease plasma thyroxin levels through interaction with transthyretin. As a consequence, plasma vitamin A and thyroxin are depleted. Both thyroxin and vitamin A are important regulators of normal epithelial differentiation and proliferation and might act in the promotion phase of carcinogenesis. In the present report, extracts of indoor and outdoor airborne particulate matter (APM), originating from different pollution sources, were tested for a possible interaction in vitro and in vivo with thyroxin and vitamin A. Results indicated that all APM extracts significantly interfered with thyroxin binding to transthyretin. In all cases, the more polluted samples inhibited binding more than the corresponding control samples. Exposure of rats to a cigarette smoke extract induced clear changes in plasma thyroxin and triiodothyronin hormone levels. Triiodothyronin levels were reduced most. Effects on plasma vitamin A levels were also found, but these were less clear. In addition a decrease in lung vitamin A levels was found, but this decrease was not statistically significant. Exposure of rats to an outdoor APM extract induced significant changes in plasma triiodothyronin levels. Effects on thyroxin levels were not significant, although the same trend was seen as for the cigarette smoke APM extract. No changes in plasma or liver vitamin A concentrations were induced by the outdoor APM extract. Lung vitamin A levels were decreased, but again this decrease was not statistically significant. Taken together, the present report shows that APM extracts have the potency to interfere with thyroid hormone metabolism both in vitro and in vivo and probably with vitamin A metabolism as well. As the thyroxin-transthyretin binding assay is rapid and sensitive, this parameter may be a useful new biomarker for assessing air quality.

1. 1. Comparative physiology may help to improve the toxicologists' ability to assess and predict toxicological risks of chemicals. 2. 2. Three main lines of approach have been distinguished, A: comparative research concerning the toxicokinetics of chemicals in different species; B: research concerning ecophysiological characteristics and C: studies aimed at the identification of biological markers that can be used to signal toxic effects in both experimental and free living populations of organisms. 3. 3. Some remarks are made on limiting conditions to be fulfilled in order to make comparative physiology valuable from a toxicological point of view.

Ecotoxicology is an interdisciplinary science that deals with the fate and adverse effects of chemicals on ecosystems. A well-balanced assessment of the adverse effects of chemical pollutants in the environment therefore, should be based on studies integrating analytical, toxicological and ecological information. Ecological and analytical information on pollutants are usually obtained from -'field' studies, while toxicological information is mostly obtained from laboratory studies with a limited number of experimental animals (rats, mice, quail, fish, Daphnia, etc.). As the abiotic and biotic environment in the field is very different from that in a laboratory study, it is impossible to predict or explain the effects in a field situation from effects observed in the laboratory. As a consequence, it is almost impossible to establish more than only an empirical relationship between concentrations in the environment or tissues and ecological effects. A large gap in the dose-effect chain still remains to be explained. The gap between field and laboratory studies can be lessened by a better understanding of the mode of fate and action of chemicals in the organism on one hand, and an improved knowledge of relevant physiological processes on the other. The integration of knowledge on physiology and mode of action of chemicals may provide relevant and sensitive parameters to monitor the toxic action of chemicals under 'field' conditions. Studying these parameters in the same species under laboratory and under field conditions may also significantly improve the validity of extrapolation of toxicity data to effects in the field. The use of more or less universal parameters, which can be measured in a wide range of species, can provide a key to the understanding why a certain species is, and another species is not, vulnerable to the toxic action of a chemical. We therefore recommend that when studying the toxicity mechanism of chemicals suspected of causing adverse ecological effects, particular attention should be given to potential interference in biochemical/endocrinological control mechanisms of physiological processes that are important for the survival of populations, such as growth, reproduction, energy utilization and osmoregulation. Such studies should be performed on laboratory animals, and on wildlife species under laboratory and semi-field conditions (Fig. 1). Sensitive toxicity parameters may thus be obtained in a retrospective way, and tested in field experiments. These parameters subsequently can be used in a prospective way as biological effect monitors in an early warning system under field conditions. This approach would also provide a better basis for extrapolation of data obtained under laboratory conditions to field situations, and of data obtained for one species to other species. We realize that such an approach requires detailed knowledge and experience in various disciplines. In many instances this cannot be met by individual researchers, but rather should be tackled by groups of researchers specialized in the different research areas. This proposal is therefore also a plea for setting up joint collaborative studies between different institutions crossing national borders.

Side-effects of cypermethrin, permethrin, and deltamethrin applications for the control of tsetse flies were studied in a sleeping sickness focus near Bouafle, Ivory Coast (West Africa). The insecticides were applied by ground-spray on the surrounding vegetation of three villages. Each village was treated with one pyrethroid. Effects on the non-target arthropod fauna were observed by means of regular and multiple sampling before and after treatment in the sprayed villages and in an untreated control village. Malaise traps and pitfall traps were used. Apparently, the three pyrethroids had markedly different side-effects. The spiderAporoptychus lamottei, however, appeared to be affected by all treatments. This species is therefore considered as an indicator for side-effects of pyrethroids used under the given circumstances. In all affected taxa, except in the proctotrupoid genusCremastobaeus, recovery was observed within two months.Muscidae were markedly affected by deltamethrin. The selectivity of this insecticide for tsetse control is discussed.

In this study the mutagenic potential of two tropical plant materials was investigated in Salmonella typhimurium strains TA98 and TA 100. The oil extract from seeds of Azadirachta indica showed no mutagenic activity in either strain with or without addition of metabolizing systems. When extracts of Phytolacca dodecandra berries were tested, only the butanol extract caused direct mutagenicity in TA98. After addition of rat liver homogenate, again only the butanol extract was positive in TA98. Addition of gut flora extract as metabolizing system generated positive effects in both the methanol extract and the butanol extract. The water extract showed only a slight positive effect, which can most probably be ascribed to the presence of histidine in the sample.

Swine liver microsomes convert the nitrofuran furazolidone into N-(4-cyano-2-oxo-3-butenylidene)-3-amino-2-oxazolidone, a reactive open-chain acrylonitrile derivative. This derivative may be trapped with such thiol-group-containing agents as glutathione and mercaptoethanol. However, this reaction is reversible; e.g., adding an excess of mercaptoethanol to an aqueous solution (pH 7.4) of the glutathione conjugate results in conversion of 43% of this compound into the mercaptoethanol conjugate. In addition, when microsomal protein is added to the glutathione conjugate or the mercaptoethanol conjugate, 36 and 44%, respectively, become covalently bound to the protein. The amount of this covalently bound radioactivity decreases again on prolonged incubation at 37 degrees C (42% disappearance within 24 hr), suggesting that the acrylonitrile derivative also reacts reversibly with thiol groups of microsomal protein. Indeed an excess of mercaptoethanol could remove covalently bound radioactivity from microsomal protein resulting in the formation of the mercaptoethanol conjugate. The reversibility of the reaction is dependent on pH, as is demonstrated for the mercaptoethanol conjugate. Below pH 2 this conjugate is stable; optimal exchange to microsomal protein is found between pH 7 and 10. At very high pH (greater than 11) no binding to protein is found, although the conjugate disappears rapidly. The mercaptoethanol conjugate exhibits mutagenic activity in the Salmonella/microsome test indicating that the acrylonitrile derivative of furazolidone also interacts with DNA.

The effects of above ground applications of pesticides in field crops on the epigeal predator fauna in the Lake Yssel polders in The Netherlands was assessed during a 20-month period by means of pitfall trapping in 17 commercially farmed fields and in one experimental field. The pesticides used during this period were the herbicides 2,4- d -amine and MCPA/MCPP and the insecticides deltamethrin, fenitrothion, and bromophos-ethyl. Deposition of the spray on the soil was determined. Bioassays were carried out in the fields in two cases. Of the taxa identified, i.e. staphylinids, carabids and spiders, the erigonid and linyphiid spiders appeared to be sensitive to the three insecticides. Long-term effects, however, could not be demonstrated. No effect was observed from the herbicide applications. Fields which were mechanically treated against weeds had a lower species diversity than chemically treated fields. Ploughing had a detrimental effect on the spiders. The effect of deltamethrin appeared to depend on weather conditions. At higher temperatures the effect was stronger than at lower temperatures, and no effects could be observed under rainy conditions. A guild of five species, i.e. Erigone atra, E. dentipalpis, Oedothorax apicatus, Meioneta rurestris and Bathyphantes gracilis , is recommended as an indicator for early detection of side-effects of insecticides on the epigeal predator fauna.

In this study the effect of polychlorinated biphenyl (PCB)-contaminated fish on plasma retinol (vitamin A) and thyroid hormone concentrations, i.e. sensitive indicators of PCB-intoxication, were investigated in the common seal Phoca vitulina. Seals fed fish from the Wadden Sea (high-level PCB contamination) had significantly lower concentrations of plasma retinol, total (TT4) and free thyroxin (FT4) and triiodothyronin (TT3) as compared to seals fed fish from the north-east Atlantic (low-level PCB contamination). The PCB-induced reduction in plasma retinol levels disappeared when seals on a Wadden Sea fish diet were subsequently fed Atlantic Ocean fish. It is suggested that reduced plasma retinol and thyroid hormone levels, which may result in an increased susceptibility to microbial infections, reproductive disorders and other pathological alterations, are critically involved in the recently reported reproductive disorders and the lethal viral infections in seals and other marine mammal populations in the Baltic, North Sea and Wadden Sea.

In three successive years from 1974 onwards observations were made on the possible side effects of helicopter applications of dieldrin and endosulfan in a number of Northern Guinea savanna zone habitats in Nigeria. In one of the areas a comparison could be made with discriminative handspray applications of endosulfan and DDT. An account is given of the acute effects on non-target species. Before, and at various intervals after, spraying a population census was made of a number of selected bird species. Certain species of fringe forest birds such as various flycatcher species appeared to be very vulnerable and either were not recorded any more or became extremely rare in the areas treated. It appears that the groundspray procedures used in Nigeria are more discriminative—and therefore less likely to cause irreversible damage—than helicopter applications at comparable dose-rate.

Several short-term mammalian test systems were used for mutagenicity testing of the organic solvent dichloromethane. The compound was negative in the forward mutation test on the HGPRT locus in Chinese hamster cells and the unscheduled DNA synthesis test in both human and hamster cells. In the test on DNA synthesis inhibition, dichloromethane caused an aspecific inhibition in both human and hamster cells, but in this test the effect did not indicate a DNA-damaging action. A weak positive effect was found in the test on sister-chromatid exchanges in hamster cells.

The acute toxicity of hexavalent chromium, Cr(VI). to rainbow trout ( Salmo gairdneri ) increased with decreasing pH in the range from 7.8 to 6.5. Morphological changes that could be associated with acute Cr(VI) poisoning at pH 7.8 were found in gills, kidney and stomach, whereas those at pH 6.5 appeared to be restricted to the gills only. At both pH values, however, similar alterations in plasma osmolality and hematocrit values of blood were found in fish surviving an exposure to acute toxic concentrations. To explain the observed effects, hydroehromate (HCrO 4 − ) and chromate (CrO 2− 4 ) were considered as the toxic species of Cr(VI). An attempt was made to calculate the relative toxicities of these ionic species from empirical toxicity relationships for weak acids in fish, as described in the literature.

We studied the effects of a complete human diet, based on mean consumption figures in The Netherlands, the heating of food, and the presence of vegetables and fruit in the diet on the drug metabolizing capacity of the rat liver and on metabolic activation of known carcinogens. Groups of five male and five female Wistar rats were given ad lib. one of six different diets for 3 months. Each diet contained 40 energy (E)% fat, 13 E% protein, 47 E% carbohydrate and 5% fibre (w/w). The diets were as follows: a control diet of semi-synthetic materials (A); a human diet of meat, bread and eggs without processing (B); diet B heated under usual household conditions (C); a diet representing a complete human meal including (summer) vegetables and fruit (D); diets consisting of winter vegetables (E) or summer vegetables (F) with fruit. Semi-synthetic components were added to diets B-F to achieve the desired composition. There were differences between male and female rats on the effects of the different diets on hepatic enzyme activity. In female rats, but not in males, ethoxyresorufin-O-deethylase activity was increased significantly (P less than 0.05) in groups C, D and E in comparison with the controls (group A). In male rats ethoxycoumarin-O-deethylase activity was enhanced in groups D, E and F, and glutathione-S-transferase was markedly induced in group F (P less than 0.01). In males, hepatic cytochrome P-450 was significantly (P less than 0.05) increased in groups B, C and E. There was no effect on aminopyrine-N-demethylase activity and almost no effect on UDP-glucuronyltransferase activity in either sex. Microsomes from rats fed heated food (C) markedly increased the mutagenicity of benzo[a]pyrene (B[a]P) in the Ames assay using Salmonella typhimurium strain TA98, in comparison with levels using microsomes from rats fed the raw food (B). Vegetables and fruit decreased B[a]P mutagenicity. All human diets except D decreased the mutagenicity of N-nitrosodimethylamine in tester strain TA100. The results indicate that the influence of components of human diets on rat-liver drug metabolism may have quite different effects on the biotransformation of carcinogens activated by different metabolic pathways.

Summary Large scale pollution of the environment by pesticides and other chemicals only started in the fifties and early sixties. Chlorinated hydrocarbon pesticides and methyl mercury compounds appeared to have a marked impact on the populations of many animal species, especially birds. It is recognized that prof. dr. H. van Genderen, who retired this month, made a most valuable contribution to the timely detection of these problems in the Netherlands. Environmental toxicologists engaged in studies to predict environmental hazards by chemicals require toxicological skill as well as insight into the structure and functioning of ecosystems.

A number of pesticides were examined for their possible porphyrinogenic properties by means of an assay procedure based on the use of primary liver cell cultures of chicken embryos. Three out of twelve compounds tested appeared to be able to induce porphyria without previous treatment of the cells with a drug enzyme inducing compound (viz. HCB, β-HCH and chlorfenvinphos). Eight out of the twelve compounds responded in the induced system (viz. HCB, β-HCH, chlorfenvinphos, temephos, azinphosmethyl, benzoyl prop-ethyl, pentachlorophenol and photomirex). No effect was found with 2,4,5-T, carbaryl, captan and trichlorfon. Enzyme induction markedly enhanced the porphyrinogenic potential of HCB, β-HCH and chlorfenvinphos. It is suggested that the measurement of the urinary porphyrin pattern may be a valuable parameter for the purpose of human biological monitoring in relation to occupational exposure to pesticides. Especially when there is some likelihood that the workers are also affected by the drug enzyme inducing properties of pesticides and/or other chemicals like drugs.

The induction of chromosome aberrations in fishes, exposed to Rhinewater, was investigated. The mudminnow, Umbra pygmaea , was chosen for this study, because of its ideal karyotype of 22 large chromosomes. Gill cells were used for chromosome studies. Fish, kept in Rhinewater for 11 days had chromosome breaks in approximately 30% of the metaphases studied. Control fish, exposed to a very good quality of untreated groundwater had breaks in about 8% of the metaphases. Several Rhinewater extracts were tested for their mutagenic potential in the Salmonella -microsome test. The fraction of aromatic compounds was found to be positive. This may indicate that one or more of the compounds present in this fraction were also responsible for the cytogenetic changes found in the fish.

The most important continuous biomonitoring systems which are presently being used for the assessment of water quality are discussed briefly. Special attention is given to their indicator value as well as to observation methods which may expand their perceptive faculty. It is concluded that a proper water quality assessment can not rely only upon the use of biological monitoring systems. They should be seconded by adequate multidetectional chemical monitoring methods.

The influence of dietary factors such as total composition, thermal processing, and the addition of vegetables and fruit on the tumour rate in rats was studied in a long-term experiment. Groups of 50 male and 50 female Wistar rats were fed one of the following diets: a semi-synthetic animal diet (A, control); diet A to which vegetables and fruit were added (B); an uncooked human diet (meat, bread and eggs) supplemented with semi-synthetic compounds (C); diet C with fried or baked products (D); a complete human diet consisting of heated products, vegetables and fruit prepared according to mean consumption figures in The Netherlands (E). The animal diets (A and B) contained 26.0 energy (E)% protein, 21.6 E% fat, 52.4 E% carbohydrate and 10.7% (w/w) fibre. The human diets contained 13.2 E% protein, 40.6 E% fat, 46.2 E% carbohydrate and 5% (w/w) fibre. The rats were fed ad lib. for 142 wk. In males and females fed human diets (C, D or E) hepatocellular vacuolization was observed. Male rats (but not female) fed the human diet had a significantly (P less than 0.02) higher incidence of epithelial tumours than those fed the animal diet. This increase was mainly due to tumours of the pituitary and thyroid. Frying and baking of food products (diet D) and the addition of vegetables and fruit (diet E) induced minor differences in tumour rate, but they were not statistically significant.

The main purpose of the present study is to develop a field monitoring system for the assessment of the possible impact on the terrestrial entomofauna caused by crop-protection measurements, including the use of insecticides and other pesticides. In the first phase of the study special attention is paid to the epigeal predator arthropod fauna, which in the area concerned mainly consists of carabid beetles and spiders. Particularly this part of the soil fauna is liable to become exposed to above ground applications of pesticides.

The carcinogenicity in male and female Wistar SSP TOX rats of organic drinking-water concentrates that are positive in the Ames test was studied at three doses. The organic mutagenic concentrates were prepared weekly from drinking-water from one location in The Netherlands by adsorption onto XAD-4/8 resins and elution with dimethylsulphoxide. The organic concentrates in dimethylsulphoxide were mixed with non-mutagenic drinking-water before exposure of the rats. Dose levels were based on multiples of expected human exposure levels. For the calculation the average human daily intake of drinking-water was taken as 2 litres for a body weight of 70 kg. There was no significant increase in tumour induction when male Wistar SSP TOX rats were exposed for 106 wk to 4.5, 14 or 40 times the expected human exposure level and females to 7,22 or 68 times the human level. The development and types of tumours were similar in the treated and control groups. The numbers of animals with tumours and of animals that died as a result of tumours in the exposed groups did not differ significantly from those in the control groups. These results suggest that these organic mutagenic drinking-water concentrates did not contain very potent carcinogens in effective concentrations.

Permethrin, cypermethrin, and decamethrin were applied in field trials to eradicate tsetse flies in the northern Guinea savanna zone in Nigeria. Aerial application of ultra-low-volume formulations of cypermethrin (150 g ai/ha) and decamethrin (30 g ai/ha) were nearly or completely successful. Biological environmental monitoring showed no effects on vertebrate populations. Acute mortality occurred in many terrestrial and aquatic arthropods, notably water beetles and crustaceans. Malaise trap catches showed significantly reduced abundance of flying insects for at least 10 days after spraying. Shrimps and mayfly larvae disappeared from river benthos samples after spraying, but were found again in samples taken 1 year later. The severe reduction in aquatic arthropod abundance may represent a hazard to fish populations through reduced food supply but this could not be assessed at the current pilot scale of spraying operations.

Fat from Aroclor 1254-treated swine was rendered and incorporated into the diets of broiler chicks for 3–4 weeks. The technical Aroclor 1254 which was fed to the swine was also mixed into control lard for comparison at dietary concentrations of 0.07–9.0 mg/kg. The swine-residue PCB seemed to have a higher proportion of strong microsomal inducers, but the technical PCB was slightly more effective in inducing ethoxy resorufin and p- nitroanisole (pNA) O- dealkylases than the swine-residue PCB. No overt signs of toxicosis were apparent and one of the diets resulted in changes in growth, relative organ weights, microsomal protein or high affinity pNA O- dealkylase . Increases in cytochrome(s) P-450 were significant only at the higher dietary concentrations (approx. 9 mg/kg) while ethoxyresorufin O- dealkylase was induced at dietary concentrations below 1 mg/kg.

Monoclonal antibodies were prepared against rat glutathione S-transferase isoenzymes 2-2 and 3-3. Three clones were capable of specifically differentiating their respective antigens from other rat isoenzymes as well as human isoenzymes, in ELISA and on Western blot. One clone produced antibodies specific for isoenzyme 2-2, and 2 hybridomas were specific for isoenzyme 3-3. Balb/c mice did not respond to immunization with glutathione S-transferase isoenzymes 1-1 and 4-4. However, an immune response was obtained in some other strains of mice, with differential H-2 haplotypes, notably CBA/BrARij mice and CBA/CaHRij-T6 mice for isoenzyme 1-1 and CBA/BrARij mice for isoenzyme 4-4, which offers perspectives for obtaining additional specific monoclonal antibodies against these glutathione S-transferases.

THERE has been considerable concern about the presence of chlorinated hydrocarbon insecticides in wild birds' eggs. The toxicology of chlorinated hydrocarbon insecticides has been evaluated1–3, using hens' eggs, and emphasis has usually been placed on the effects on hatching. In the experiments of Dunachie and Fletcher, hatchability was not affected even by very large doses (for example, 200 p.p.m, of dieldrin).

In a comparative 60-day oral toxicity test (400 ppm) in chickens, three 60%-chlorinated commercial polychlorinated biphenyl (PCB) preparations were used: compound I (Phenoclor DP6), compound II (Clophen A60), and compound III (Aroclor 1260). Using mortality, mean survival time, mean weight, and pathological observations (hydropericardium, abdominal, and subcutaneous edema and centrolobular liver necrosis) as parameters, a significant difference in toxicity was found between the compounds: compounds I and II showed the highest, compound III the lowest, toxicity. Microscopically centrolobular liver necrosis was found in chicks fed compounds I and II. Atrophy of the spleen was found in all test groups. Chemical porphyria was found as a general PCB effect: increased fecal excretion of coproporphyrin and protoporphyrin and fluorescence of tissues occurred in all test groups. Additional experiments with compound I (2000 ppm) in Japanese quail and rats confirmed the porphyrogenic action. Gas chromatographic analyses of liver and brain of dead chicks gave PCB levels that varied between 120 and 2900 ppm. The relationship of hydropericardium (chick edema) and liver necrosis to the differences in toxicity observed between the technical PCB mixtures is discussed.

Polychlorinated biphenyls are widely used in industry, and they are now making an appearance in the world's wildlife. Besides being a hazard in their own right, they interfere with the detection of organochlorine insecticides such as DDT.

In a previous study (Vos & Koeman, Toxic. appl. Pharmac. 1970. In press) a significant difference in toxicity was found between three commercial polychlorinated biphenyl (PCB) preparations, high mortality, liver necrosis and chick oedema-like lesions being associated with only two of the compounds tested. In the present study, column and gas chromatography demonstrated the presence of polar compounds in the 25% diethyl ether fraction of each of these two PCB preparations, and a chick-embryo assay confirmed the high toxicity of this fraction. The polar compounds were not found in the corresponding fraction of the third PCB preparation; neither did this preparation have a significantly toxic effect on the chick embryo. Subsequent mass spectrometric and microcoulometric analyses indicated the identity of the polar compounds, which included tetra- and pentachlorodibenzofuran. The significance of these impurities is discussed. It is concluded that evaluation of animal tests on PCB is made extremely difficult by the presence of chlorinated dibenzofurans in some commercial PCB preparations.

The research described in this thesis was aimed at developing an in vitro cell-based reporter gene system applicable to the detection of the illegal use of androgenic growth promoters in cattle, and the presence of potential endocrine disrupters present in surface waters and interfering with androgenic action. The system is based on a luciferase reporter gene placed under transcriptional control of an authenticated androgen-responsive element (ARE) and an endogenously expressed androgen receptor. This system allows for the integration of the effects of certain modulators of androgenic signal transduction. A second important goal of the research was to gain insight into the mechanisms underlying enhanced growth promotion by mixtures of androgenic and estrogenic compounds. The use of such mixtures, which results in activation and subsequent interaction of multiple steroid receptors, is occasionally observed in illegal hormonal treatments of cattle.When applied to the screening of calf urine samples for anabolic androgens, the developed AR-LUX assay was able to identify androgen-treated animals with similar results as obtained by standard GC-MS analysis. However, both techniques should be regarded as complementary rather than interchangeable screening tools. Liquid samples confiscated at cattle farms outside theNetherlandswere found to generate a very strong response in the AR-LUX assay despite the fact that GC-MS analysis did not detect the presence of any anabolic compounds. Possibly, the samples contained a mixture of conventional androgenic compounds, each at undetectably low amounts and/or (novel) unknown compounds not tested for by GC-MS. These results emphasize the additional value of the developed AR-LUX assay.Also, the AR-LUX assay was used to determine the androgenic activity of a number of aquatic environmental samples. A number of these samples were found to contain androgenic activity at varying concentrations. Interestingly, in 2 samples containing androgens, enhancing interactive mixture effects were observed, which were probably due to interactions by estrogenic compounds and estrogen receptor activation.Our research furthermore indicates that certain established progestagens are able to activate ARE-mediated luciferase expression via progesterone receptors; we hypothesise preferentially through the progesterone receptor-α isoform. This indicates that androgen reporter assays based on the activation of the androgen receptor alone rather than on activation of a response element might produce results quite different from those observed in assay systems featuring multiple steroid receptors. This further emphasizes the notion that the AR-LUX assay is not merely detecting activation of the androgen receptor by androgens, but also allows for the detection of other androgenic substances that regulate gene expression via alternative pathways leading to activation of an established androgen response element.