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To derive an acute TTC threshold, the correlation between Allowable Daily Intakes (ADIs, chronic values) and Acute Reference Doses (ARfDs) of pesticides evaluated in the EU was investigated and their distributions were compared. The correlation between ARfDs and ADIs was significant (p = 0.01), but weak (r(2) = 0.051). Consequently, using this approach to derive acute TTC values does not seem valid. Therefore, the distributions of ARfDs and ADIs were compared directly, in order to extrapolate from chronic to acute TTC values. This comparison made for the combined Cramer structural class II and III pesticides showed a ratio ARfD/ADI of approximately 3 at the fifth percentile of the distributions. Based on these results, it is justified to propose a TTC for acute effects for Cramer III substances by multiplying the Cramer class III TTC threshold of 90 μg/person/day with a factor 3. This leads to an acute TTC threshold based on the Munro dataset for Cramer class III substances of 270 μg/person/day.

Precision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an efficient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies confirmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was confirmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices. Copyright (C) 2000 Elsevier Science Ltd. Chemicals/CAS: Adenosine Triphosphate, 56-65-5; Dinitrochlorobenzene, 97-00-7; Formazans; Glutathione Transferase, EC 2.5.1.18; Glutathione, 70-18-8; Lactate Dehydrogenase, EC 1.1.1.27; MTT formazan, 23305-68-2; Proteins; Testosterone, 57-85-2; Tetrazolium Salts; Urea, 57-13-6

The susceptibility to nephrotoxins of freshly isolated porcine proximal tubular cells (PPTC) and cultured PPTC in suspension were compared, with a view to using PPTC as in vitro models for studying nephrotoxicity. In a previous paper, we described how, in freshly isolated PPTC, well-known nephrotoxins such as mercury (II) chloride, cisplatin, p-aminophenol and halogenated hydrocarbons caused a dose-dependent decrease in the viability and mitochondrial membrane potential of the PPTC. In this paper, we show that suspensions of cultured PPTC, harvested by trypsinisation, are less susceptible to nephrotoxins, possibly due to the synthesis of extracellular matrix components. PPTC in primary culture are suitable for relatively long-term nephrotoxicity studies. This was demonstrated by incubation with mercury (II) chloride for 24 hours, resulting in a dose-dependent loss of viability. Freshly isolated PPTC can be cryopreserved by computer-controlled freezing. The cryopreserved PPTC displayed an increased susceptibility to mercury (II) chloride and a decreased susceptibility to cisplatin and 1,1-dichloro-2,2-difluoroethylene-cysteine, the toxicity of the latter indicating that the renal enzyme β-lyase remains active during cryopreservation.

To test for potential estrogenic activity of plant stanols and plant stanol esters, two short-term tests were performed. These were the E-screen test, which measures a substance's ability to induce proliferation of estrogen-responsive human breast adenocarcinoma (MCF-7) cells in culture, and an in vivo test, which measures uterotrophic activity in immature female rats fed the test substance. Four samples of vegetable oil-derived stanols (containing 88-99% stanols) were tested in the E-screen test, and one sample of wood-derived and one of vegetable oil-derived stanol fatty acid esters were tested in the in vivo test. In the E-screen test, the positive control substance, 17beta-estradiol, at 100 pM, produced a statistically significant, 11.6-fold increase in cell proliferation, as measured by sulforhodamine B staining. None of the stanol preparations produced any increase in cell proliferation when tested at 1, 10, and 100 microM. The highest dose of each stanol sample was associated with microscopic evidence of cytotoxicity and crystalline precipitation in the culture dishes. In the in vivo test, the positive control compound, diethylstilbestrol, produced a significant, dose-related increase in absolute and relative uterus weight in young female rats (17 days old at the start of treatment) fed the compound at 5, 10, and 20 ppb in the diet for 4 days. Neither of the two stanol ester preparations caused any significant change in absolute or relative uterus weight when fed at a concentration of 8.3% in the diet for 4 days. Thus, under the conditions of testing used, neither the free stanols nor the stanol fatty acid ester preparations showed evidence of estrogenic or uterotrophic activity.

The present paper reports about an immunocytochemical inventory of the cell types involved in the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to a DNA methylating metabolite. The formation and distribution of the methylated DNA bases O6-methylguanine (O6-meGua) and 7-methylguanine (7-MeGua) were studied in respiratory tissues, oesophagus, liver, kidneys, pancreas, small intestine, colon and prostate of rat, mouse and hamster 6 h after treatment with a single dose of 30 mg NNK/kg. The tissue- and cell-specific distribution of O6-meGua- and 7-meGua-specific nuclear staining showed the same patterns and were remarkably similar in rat, mouse and hamster in spite of the diverging spectra of NNK-induced tumours in these species. In nasal tissue, a target for NNK-induced tumourigenesis in rat and hamster, but not in mouse, adduct-specific nuclear staining was observed in all three species in sustentacular cells, Bowman glands, respiratory epithelial cells and serous glands. Both methylated DNA bases were also observed in basal cells of the olfactory epithelium of rat and (occasionally) hamster, but not in those of the mouse. In the trachea, a target for NNK-induced tumourigenesis in hamster only, substantial adduct-specific nuclear staining was found in basal epithelial and glandular cells of the hamster; in the same cells of rat and mouse only a weak nuclear staining was found. In the lung, a common target for NNK-induced tumourigenesis, the formation of O6-meGua and 7-meGua was restricted predominantly to bronchial and proximal bronchiolar epithelium. Nuclear staining in the rat was occasionally found in alveolar cells and was also observed in hepatocytes. In the three species investigated, O6-meGua- and 7-MeGua-specific nuclear staining was found in target and non-target tissues. Apparently, and in analogy with results obtained in other studies, the species-specific organotropy for tumour formation of NNK is not exclusively determined by DNA methylation. Expanding methylation data with literature data on factors considered to be involved in tumour formation, namely proliferation, toxicity and DNA repair among others, still did not lead to a satisfactory explanation for the species-specific organotropy observed. Additional factors (yet to be identified), need to be taken into account in order to explain (and predict) tumourigenic effects induced by monofunctional methylating agents.

Male Wistar rats were exposed for 4 weeks, 5 days a week, to 0 (controls), 5 or 10 ppm formaldehyde continuously (8 hours a day), or to 10 or 20 ppm formaldehyde interruptedly (eight 30 min exposure periods separated by 30 min non-exposure periods). Histopathology and cell proliferation studies indicated that under the conditions of exposure used, concentration rather than the total dose of formaldehyde determined the severity of the cytotoxic effects on the nasal epithelium.

Male Wistar rats were exposed for 13 weeks, 5 days a week to 0 (controls), 1 or 2 ppm formaldehyde continuously (8 h a day), or to 2 or 4 ppm formaldehyde interruptedly (eight 30-min exposure periods separated by 30-min non-exposure periods a day). Histopathological changes were only found in the nose of animals (interruptedly) exposed to 4 ppm formaldehyde and comprised an increased degree and incidence of disarrangement and squamous metaplasia accompanied by basal cell hyperplasia and occasionally by keratinization of the respiratory epithelium. Two ppm formaldehyde was the non-toxic effect level. Cell proliferation studies demonstrated a slightly higher cell turnover of the nasal respiratory epithelium exposed (interruptedly) to 4 ppm formaldehyde than in controls. It was concluded that under the conditions of repeated exposure to marginally cytotoxic concentrations during a period of 13 weeks the exposure concentration rather than the total 'dose' (= concentration x exposure time) determined the severity of the cytotoxic effects of formaldehyde on the nasal epithelium.

Cyanoketone administered via the food (0.1, 0.2 and 2 mg/g) for 8 weeks from the first feeding (day 46 after fertilization) or via the aquarium water (3 and 30 mg/100 l) for 4 weeks from day 41 does not influence the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the interstitial cells of the gonads or interrenal cells of juvenile trout in vivo. However, the number of 3 beta-HSD-positive interrenal cells was strongly increased by administration of the highest dose of cyanoketone via both routes. These high doses furthermore affect the sex ratio in favor of males. It is concluded that interrenal tissue is responsible for the masculinizing effect of cyanoketone via increased production of androgens and/or corticosteroids. Cyanoketone at concentrations of 0.01 to 100 micrograms/ml causes a dose-response inhibition of 3 beta-HSD activity in the interrenal cells, when the substance is administered to an incubation medium for demonstration of this enzyme in tissue sections. The controversial in-vivo and in-vitro effects of cyanoketone on 3 beta-HSD activity are discussed.