Genome-wide expression screening in the cardiac embryonic stem cell test shows additional differentiation routes that are regulated by morpholines and piperidines.

The cardiac embryonic stem cell test (ESTc) is a well-studied non-animal alternative test method based on cardiac cell differentiation inhibition as a measure for developmental toxicity of tested chemicals. In the ESTc, a heterogenic cell population is generated besides cardiomyocytes. Using the full biological domain of ESTc may improve the sensitivity of the test system, possibly broadening the range of chemicals for which developmental effects can be detected in the test. In order to improve our knowledge of the biological and chemical applicability domains of the ESTc, we applied a hypothesis-generating data-driven approach on control samples as follows. A genome-wide expression screening was performed, using Next Generation Sequencing (NGS), to map the range of developmental pathways in the ESTc and to search for a predictive embryotoxicity biomarker profile, instead of the conventional read-out of beating cardiomyocytes. The detected developmental pathways included circulatory system development, skeletal system development, heart development, muscle and organ tissue development, and nervous system and cell development. Two pesticidal chemical classes, the morpholines and piperidines, were assessed for perturbation of differentiation in the ESTc using NGS. In addition to the anticipated impact on cardiomyocyte differentiation, the other developmental pathways were also regulated, in a concentration-response fashion. Despite the structural differences between the morpholine and piperidine pairs, their gene expression effect patterns were largely comparable. In addition, some chemical-specific gene regulation was also observed, which may help with future mechanistic understanding of specific effects with individual test compounds. These similar and unique regulations of gene expression profiles by the test compounds, adds to our knowledge of the chemical applicability domain, specificity and sensitivity of the ESTc. Knowledge of both the biological and chemical applicability domain contributes to the optimal placement of ESTc in test batteries and in Integrated Approaches to Testing and Assessment (IATA).

Isoflucypram: Combining in vivo and NAMs data in a weight of evidence approach to demonstrate the human non-relevance of the mode of action leading to the subtle thyroid effects observed in the rat.

Isoflucypram (ISY) is a new cereal fungicide with an overall favorable toxicity profile. As the thyroid was identified as a target organ only in the rat, following repeat dosing; short term in vivo (rat) and in vitro mechanistic studies were conducted to substantiate the thyroid changes as being secondary to liver enzyme induction via PXR/CAR activation and to determine the human non-relevance of the thyroid effects. The in vivo studies established ISY as a weak prototypical hepatic PXR/CAR enzyme inducer (P450 and T4-UDP-glucuronosyltransferase (UDPGT-T4) activities), with the induction being associated with increased liver weight/hepatocellular hypertrophy/proliferation. Thyroid effects (minimal follicular cell hypertrophy/proliferation, slight, statistically significantly increased thyroid stimulating hormone) occurred at doses where liver stimulation was already established. Direct thyroid effects (in vitro thyroid peroxidase and sodium iodide symporter inhibition) were excluded. Marked quantitative species differences were identified when comparing rat and human hepatic enzyme activities in vitro, particularly for UDPGT-T4. Specifically, basal UDPGT-T4 was 4-fold lower in human compared to rat hepatocytes. In addition, UDPGT-T4 was induced in vitro in rat but not in human hepatocytes following ISY treatment. Overall, the weight of evidence supports a liver mediated mode of action for the isoflucypram-induced slight thyroid changes in the rat as well as the human non-relevance of these findings.

Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Molecular neural crest cell markers enable discrimination of organophosphates in the murine cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) originally used the differentiation of beating cardiomyocytes for embryotoxicity screenings of compounds. However, the ESTc consists of a heterogeneous cell population, including neural crest (NC) cells, which are important contributors to heart development in vivo. Molecular markers for NC cells were investigated to explore if this approach improved discrimination between structurally related chemicals, using the three organophosphates (OP): chlorpyrifos (CPF), malathion (MLT), and triphenyl phosphate (TPP). To decrease the test duration and to improve the objective quantification of the assay read-out, gene transcript biomarkers were measured on study day 4 instead of the traditional cardiomyocyte beating assessment at day 10. Gene expression profiling and immunocytochemistry were performed using markers for pluripotency, proliferation and cardiomyocyte and NC differentiation. Cell proliferation was also assessed by measurements of embryoid body (EB) size and total protein quantification (day 7). Exposure to the OPs resulted in similar patterns of inhibition of beating cardiomyocyte differentiation and of myosin protein expression on day 10. However, these three chemically related compounds induced distinctive effects on NC cell differentiation, indicated by changes in expression levels of the NC precursor (Msx2), NC marker (Ap2α), and epithelial to mesenchymal transition (EMT; Snai2) gene transcripts. This study shows that investigating NC markers can provide added value for ESTc outcome profiling and may enhance the applicability of this assay for the screening of structurally related test chemicals.

Functional organisation of the endomembrane network in the digestive gland of the Venus flytrap: revisiting an old story with a new microscopy toolbox.

Up-to-date imaging approaches were used to address the spatiotemporal organisation of the endomembrane system in secretory cells of Dionaea muscipula. Different 'slice and view' methodologies were performed on resin-embedded samples to finally achieve a 3D reconstruction of the cell architecture, using ultrastructural tomography, array tomography, serial block face-scanning electron microscopy (SBF-SEM), correlation, and volume rendering at the light microscopy level. Observations of cryo-fixed samples by high-pressure freezing revealed changes of the endomembrane system that occur after trap activation and prey digestion. They provide evidence for an original strategy that adapts the secretory machinery to a specific and unique case of stimulated exocytosis in plant cells. A first secretion peak is part of a rapid response to deliver digestive fluids to the cell surface, which delivers the needed stock of digestive materials 'on site'. The second peak of activity could then be associated with the reconstruction of the Golgi apparatus (GA), endoplasmic reticulum (ER) and vacuolar machinery, in order to prepare for a subsequent round of prey capture. Tubular continuum between ER and Golgi stacks observed on ZIO-impregnated tissues may correspond to an efficient transfer mechanism for lipids and/or proteins, especially for use in rapidly resetting the molecular GA machinery. The occurrence of one vacuolar continuum may permit continuous adjustment of cell homeostasy. The subcellular features of the secretory cells of Dionaea muscipula outline key innovations in the organisation of plant cell compartmentalisation that are used to cope with specific cell needs such as the full use of the GA as a protein factory, and the ability to create protein reservoirs in the periplasmic space. Shape-derived forces of the pleiomorphic vacuole may act as signals to accompany the sorting and entering flows of the cell.

Low dose evaluation of the antiandrogen flutamide following a Mode of Action approach.

The dose-response characterization of endocrine mediated toxicity is an on-going debate which is controversial when exploring the nature of the dose-response curve and the effect at the low-end of the curve. To contribute to this debate we have assessed the effects of a wide range of dose levels of the antiandrogen flutamide (FLU) on 7-week male Wistar rats. FLU was administered by oral gavage at doses of 0, 0.001, 0.01, 0.1, 1 and 10mg/kg/day for 28 days. To evaluate the reproducibility, the study was performed 3 times. The molecular initiating event (MIE; AR antagonism), the key events (LH increase, Leydig cell proliferation and hyperplasia increases) and associated events involved in the mode of action (MOA) of FLU induced testicular toxicity were characterized to address the dose response concordance. Results showed no effects at low doses (<0.1mg/kg/day) for the different key events studied. The histopathological changes (Leydig cell hyperplasia) observed at 1 and 10mg/kg/day were associated with an increase in steroidogenesis gene expression in the testis from 1mg/kg/day, as well as an increase in testosterone blood level at 10mg/kg/day. Each key event dose-response was in good concordance with the MOA of FLU on the testis. From the available results, only monotonic dose-response curves were observed for the MIE, the key events, associated events and in effects observed in other sex related tissues. All the results, so far, show that the reference endocrine disruptor FLU induces threshold effects in a standard 28-day toxicity study on adult male rats.

Thyroid tumor formation in the male mouse induced by fluopyram is mediated by activation of hepatic CAR/PXR nuclear receptors.

Fluopyram, a broad spectrum fungicide, caused an increased incidence of thyroid follicular cell (TFC) adenomas in males at the highest dose evaluated (750ppm equating to 105mg/kg/day) in the mouse oncogenicity study. A series of short-term mechanistic studies were conducted in the male mouse to characterize the mode of action (MOA) for the thyroid tumor formation and to determine if No Observed Effect Levels (NOELs) exist for each key event identified. The proposed MOA consists of an initial effect on the liver by activating the constitutive androstane (Car) and pregnane X (Pxr) nuclear receptors causing increased elimination of thyroid hormones followed by an increased secretion of thyroid stimulating hormone (TSH). This change in TSH secretion results in an increase of TFC proliferation which leads to hyperplasia and eventually adenomas after chronic exposure. Car/Pxr nuclear receptors were shown to be activated as indicated by increased activity of specific Phase I enzymes (PROD and BROD, respectively). Furthermore, evidence of increased T4 metabolism was provided by the induction of phase II enzymes known to preferentially use T4 as a substrate. Additional support for the proposed MOA was provided by demonstrating increased Tsh ß transcripts in the pituitary gland. Finally, increased TFC proliferation was observed after 28days of treatment. In these dose-response studies, clear NOELs were established for phase 2 liver enzyme activities, TSH changes and TFC proliferation. Furthermore, compelling evidence for Car/Pxr activation being the molecular initiating event for these thyroid tumors was provided by the absence of the sequential key events responsible for the TCF tumors in Car/Pxr KO mice when exposed to fluopyram. In conclusion, fluopyram thyroid toxicity is mediated by activation of hepatic Car/Pxr receptors and shows a threshold dependent MOA.

Liver tumor formation in female rat induced by fluopyram is mediated by CAR/PXR nuclear receptor activation.

Fluopyram is a broad spectrum fungicide targeting plant pathogenic fungi (eg. white dot, black mold, botrytis). During the general toxicity evaluation of fluopyram in rodents, the liver was identified as a target organ (hepatomegaly and liver hypertrophy were observed in all studies). At the end of the guideline carcinogenicity study, an increased incidence of hepatocellular adenomas and carcinomas was observed in female Wistar rats following exposure to the highest fluopyram dose evaluated (1500ppm). Short-term mechanistic studies (3, 7 or 28days of exposure) were conducted in the female rat to identify the initial key events responsible for the tumor formation and to establish thresholds for each of the early hepatic changes. Increased expression of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) inducible genes was recorded after each exposure period. Further confirmation of CAR/PXR activation was provided by increased activity of specific Phase I enzymes (PROD/BROD respectively). Increased hepatocellular proliferation (measured by Ki67) was observed after each exposure period with the greatest proliferative response occurring after 3days of treatment. In these studies, dose responses and clear thresholds were established for gene expression, enzyme activity and cell proliferation. Furthermore, these early hepatic changes were shown to be reversible following compound withdrawal. Other modes of action for liver tumor formation such as DNA damage, cytotoxicity and peroxisome proliferation were excluded during the investigations. In conclusion, fluopyram is a threshold carcinogen and the resultant hepatocellular carcinomas in the female rat are due to hepatocellular proliferation mediated by CAR/PXR activation.

The screening of everyday life chemicals in validated assays targeting the pituitary-gonadal axis.

Ten structurally diverse chemicals (vitamins C, B9, B6, B3, sucrose, caffeine, gingerol, xanthan gum, paracetamol, ibuprofen) deemed intrinsic to modern life but not considered as endocrine active, were tested in vitro using the human estrogen receptor transcriptional activation (hERTa) and the H295R steroidogenesis assays. All were inactive in the hERTa assay but paracetamol, gingerol, caffeine and vitamin C affected steroidogenesis in vitro from 250, 25, 500 and 750 µM respectively. One molecule, caffeine, was further tested in rat pubertal assays at the tumorigenic dose-level and at dose-levels relevant for human consumption. In females pubertal parameters (vaginal opening, estrus cycle), ovarian weight and Fsh and prolactin transcript levels were affected. In males, plasma progesterone levels and prostate and seminal vesicle weights were affected. Although the current regulatory focus is synthetic chemicals that can cause adverse effects on the hypothalamus-pituitary-gonadal axis, our data infer that the range of natural chemicals with the potential to affect this axis may be extensive and is probably overlooked. Thus, to avoid regulation of an overwhelming number of chemicals, a weight of evidence approach, combining hazard identification and characterization with exposure considerations, is needed to identify those chemicals of real regulatory concern.

A novel method for measuring aromatase activity in tissue samples by determining estradiol concentrations.

Increasing scrutiny of endocrine disrupters has led to changes to European pesticide and biocide legislation and to the introduction of the Endocrine Disrupter Screening Program by the US EPA. One element of endocrine disrupter identification is to determine its effects on aromatase, but most available assays are limited as they depend on tritiated water production to indicate enzyme activity. Whilst acceptable for determining aromatase effects using a cell-free approach, this method is unreliable for cell or tissue-based investigations as other cytochrome P-450 isoenzyme activities can similarly produce tritiated water and consequently confound interpretation of the aromatase data. To address this lack of specificity an assay directly measuring the final estrogen product by incubating rat tissue protein with testosterone and measuring the resultant estradiol concentration was developed. Using this approach we demonstrated marked increases in enzyme activity in pregnant rat ovary samples and dose-related inhibitions when incubating non-pregnant rat ovary samples with known aromatase inhibitors. Hepatic aromatase activity was investigated using our method and by tritiated water production with microsomes from rats dosed with the antiandrogen 1,1-dichloro-2,2-bis(4 chlorophenyl)ethane. Additional cytochrome P-450s were also measured. Treatment-related increased tritiated water production and general hepatic enzyme activity were recorded but estradiol was not increased, indicating that the increased tritiated water was due to general enzyme activity and not aromatase activity. A simple and specific method has been developed that can detect aromatase inhibition and induction, which when applied to tissue samples, provides a means of generating relevant animal data concerning chemical effects on the aromatase enzyme.

Inter-laboratory comparisons of assessment of the allergenic potential of proteins in mice.

Assessment of the potential allergenicity of novel proteins, including those expressed in genetically modified plants, is an important issue. In previous studies, we have shown that the IgE measurement induced by systemic exposure of BALB/c mice to a range of proteins correlates broadly with what is known of their allergenic potential in humans. The approach used a homologous passive cutaneous anaphylaxis (PCA) assay that reflects IgE-dependent biological activity and is of sufficient sensitivity to detect IgE production in the absence of adjuvant. In previous studies, the immunization phase was conducted independently in two separate facilities, and the subsequent analytical work (PCA) conducted in a single facility. The purpose here was to further evaluate the transferability of this approach. To this end, BALB/c mice were exposed to a range of doses of peanut agglutinin or ovalbumin, allergenic proteins of peanut and hen's egg, respectively, in two independent laboratories. Serial doubling dilutions of serum pooled for each treatment group were analyzed for specific IgE. At higher doses of allergen very similar, or identical, IgE titers were achieved in both laboratories, although at lower doses, responses were somewhat more variable. These data demonstrate that, although technically demanding, the measurement of protein allergen-induced IgE antibody production in mice using PCA is relatively robust and is transferable and reproducible between laboratories. This approach may provide a useful tool for the safety assessment of novel proteins and suggests that continued evaluation of the approach with a wider range of protein allergens and non-sensitising proteins is justified.

The mitochondrial-dependent pathway is chronically affected in testicular germ cell death in adult rats exposed in utero to anti-androgens.

In utero exposure to exogenous anti-androgenic compounds induces a wide range of abnormalities of the reproductive system, including hypospermatogenesis, cryptorchidism and hypospadias. By using rats exposed in utero to the anti-androgenic compound flutamide (0.4, 2 or 10 mg/kg per day), it has been shown that hypospermatogenesis in adult testes could be related to (i) a long-term apoptosis in germ cells but not in somatic Leydig and Sertoli cells as evidenced by the TUNEL approach and (ii) alterations in the mRNA and protein expression of pro- (Bax, Bak, Bid) and anti-apoptotic (Bcl-2, Bcl-w) members of the Bcl-2 family. Indeed, the number of apoptotic germ cells increased with the dose of flutamide administered and the apoptotic germ cells were mainly detected at androgen-dependent stages VII-VIII. Moreover, for the Bcl-2-related proteins that were expressed mainly in the germ cells, a decrease in the levels of anti-apoptotic peptides Bcl-w (60%, P=0.003) and Bcl-2 (90%, P=0.0001) was observed at 2 mg/kg per day flutamide and an increase in levels of the pro-apoptotic Bax (2.3-fold, P=0.0004) was detected at 10 mg/kg per day. In contrast, the levels of pro-apoptotic peptide Bak that was mainly expressed in somatic cells decreased (70%, P=0.0008) at 10 mg/kg per day. Such alterations in Bcl-2-related peptides occurred mainly at the protein level except for Bcl-2 (72%, P=0.0001) and Bak (43%, P=00002) transcripts. Together, these results showed that the apoptosis observed in adult germ cells from rats exposed in utero to flutamide may result from a long-term alteration in the balance between pro- and anti-apoptotic Bcl-2-related molecules in favour of pro-apoptotic proteins. These data further supported the concept of an androgen-dependent fetal programming that is in relation with an alteration of the expression of Bcl-2-related genes/proteins promoting apoptosis in testicular germ cells of adult rats with fetal androgen disruption.

Optimisation of cell-based assays for medium throughput screening of oxidative stress.

Identification and development of cell-based assays adapted to medium throughput screening requirements is important when screening chemicals for their potential toxic properties. We describe here rapid fluorometer-based and spectrophotometer-based microplate assays which allow for the evaluation of oxidative stress in hepatocyte cell cultures by measurement of three markers: production of hydroperoxide assessed by the DCFH-DA probe, cellular antioxidant status by measurement of reduced glutathione and glutathione peroxidase activity and cytotoxicity and mitochondrial damage by evaluation of the mitochondrial transmembrane potential with rhodamine 123 fluorescent dye. The assays described here are rapid, simple and inexpensive, which are desirable when setting up screening assays. This system should be useful in selecting candidate compounds during the pre-development phase of agrochemicals or pharmaceuticals. It should also be of interest for retrospective and explanatory studies of mechanisms underlying toxicity observed in vivo.

Time course of cytochromes P450 decline during rat hepatocyte isolation and culture: effect of L-NAME.

The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte microsomal cytochrome (CYP)-dependent monooxygenase activities: CYP 1A1/2-, 2B1/2-, 3A1/2- and 2E1-dependent activities in microsomes from rat hepatocytes after isolation were about 60% of that of liver microsomes, and CYP 4A1-dependent activity was equivalent to liver microsomes. In contrast, the microsomal protein content of the various CYP isoforms was not affected by hepatocyte isolation. This is in accordance with the hypothesis of CYP inactivation during the process of hepatocyte isolation by collagenase digestion. L-NAME (1 mM) was found unable to protect from the decline of CYP-dependent monooxygenase activities following hepatocyte isolation. It is possible that the decrease in glutathione peroxidase activity observed in the presence of L-NAME, namely depression of defense against peroxynitrite, could counteract the beneficial effect of L-NAME on nitric oxide synthesis inhibition. The present work also shows that L-NAME could not avoid the progressive, isoform-specific, most probably turnover-related, decline of CYP proteins and related monooxygenase activities in cultured hepatocytes. Dysregulations in the mechanisms of CYP expression in rat hepatocyte cultures, presently unknown but nitric oxide independent, thus appear to occur in cultured rat hepatocytes.

Tumorigenic potential of carbaryl in the heterozygous p53 knockout mouse model.

The heterozygous p53 knockout mouse model was used to assess whether vascular tumors noted in a 2-year carcinogenicity study in CD-1 mice with carbaryl were induced through a genotoxic mechanism. This knockout mouse model was selected for carbaryl because of the high sensitivity of this model to genotoxic events and its low spontaneous incidence of tumors until 9-12 months of age. Carbaryl was administered continuously via the diet to groups of 20 male heterozygous p53 knockout mice at concentrations of 0, 10, 30, 100, 300, 1000 and 4000 ppm for 180 days. Histopathological examinations revealed no evidence of carbaryl-induced neoplasms of any type. In particular, no neoplastic or preneoplastic changes were noted in the vascular tissue of any of the organs examined. Only neoplasms, recognized as those that occur spontaneously in untreated mice of this strain, were sporadically observed in a few animals from the intermediate dose groups with no evidence of a dose- or treatment-related effect. Therefore, under the conditions of this study, the no-observed-effect level (NOEL) of carbaryl for neoplastic changes in male mice was 4000 ppm (around 716 mg/kg body weight/day). We conclude: (1) carbaryl does not appear to be a genotoxic carcinogen at least in male mice; (2) if the vascular tumors observed in the CD-1 mice are treatment-related, they could have been induced by a non-genotoxic mechanism; (3) the response in transgenic animals may provide useful complementary results to better assess carbaryl's potential genotoxic hazard to humans.

Diagnóstico das paralisias flácidas. Relato de três casos

O artigo não apresenta resumo.

Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes.

We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or three-dimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)- and UDP-glucuronosyl transferase (UGT)- dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix-cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT- and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.

Spontaneous nitric oxide in hepatocyte monolayers and inhibition of compound-induced apoptosis.

Primary cultures of hepatocytes are a widely used in vitro model for biochemical research. Following isolation, hepatocytes produce large amounts of nitric oxide (NO), which is known to have both pro- and anti-apoptotic effects in hepatocytes in vivo and in vitro. Previous work has not determined the effect of these increased levels of NO on the response of hepatocytes to apoptotic stimuli. Here we report that levels of nitrites are elevated in hepatocyte monolayers from 24 h onwards. Addition of the inducible nitric oxide synthase (iNOS) inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), to the medium inhibited this increase in nitrites. These results indicate that the increase in nitrite is most likely due to the formation of NO. Elevated nitrite levels had no effect either on basal levels of apoptosis or on ATP and GSH. Apoptosis was induced by transforming growth factor beta-1 (TGFbeta-1) or glycochenodeoxycholate (GCDC). Both compounds caused moderate hepatocyte apoptosis; however, addition of L-NAME prior to exposure significantly increased the level of apoptosis observed with the two compounds. Both TGFbeta-1 and GCDC had no effect on hepatocyte ATP or GSH levels; however, as a consequence of secondary necrosis, TGFbeta-1 exposure significantly increased levels of lactate dehydrogenase (LDH) leakage. These findings indicate that the increased levels of NO associated with the culture of hepatocytes have an inhibitory effect on compound-induced apoptosis in the cells.

The challenge posed by endocrine-disrupting chemicals.

Rapid regulatory developments in the area of environmental endocrine disruption present a series of potential problems that are identified and illustrated with examples taken from the recent literature. A list of priorities is provided, including the need for additional epidemiological and wildlife studies, the derivation of a coordinated testing strategy, agreement on the toxicities expected of endocrine disrupting agents, and acceptance that whole animal assays will be uniquely critical in this area of toxicology. The intrinsic difficulty of attempting to simultaneously study all aspects of endocrine disruption indicates the need to reduce the scope of the problem, which can be achieved by first studying toxicities mediated by sex hormone receptors.

Induction of cytochrome P450 and peroxisomal enzymes by clofibric acid in vivo and in vitro.

We have analysed the induction of microsomal and peroxisomal proteins and their RNAs after treatment of hepatocytes with the peroxisome proliferator, clofibric acid, in vitro and in vivo. After treatment of hepatocytes with 1 mM clofibric acid for 4 days, P450 4A1 RNA is induced 500-fold, and acyl-CoA oxidase and P450 2B1 280-fold, relative to control cultures. These RNAs are detectably induced after administration of 25 microM clofibric acid, and show a similar induction response with increasing doses of clofibric acid. Western blot analysis of the P450 4A and bifunctional enzyme (BFE) proteins showed that both were induced in parallel with increasing doses of clofibric acid, over a range of 25 microM-1 mM. The distribution of the induced proteins was examined by immunocytochemistry. Increasing doses of clofibric acid led to an increase in the average intensity of staining for both proteins throughout the hepatocyte population. There was, however, a graded variation between hepatocytes in the intensity of staining, both for P450 4A and BFE proteins. The heterogeneity in response of the hepatocyte population in vitro may be related to differential sensitivity of hepatocytes to induction in vivo. Therefore, rats were dosed with 0, 50 or 300 mg/kg of clofibric acid for 4 days by gavage, and the livers were examined by immunocytochemistry. After 50 mg/kg of clofibric acid, both P450 4A and BFE were induced mainly in zones 3 and 2 of the liver acinus. However, after 300 mg/kg of clofibric acid, staining for both proteins was strong and homogenous throughout the liver acinus. Thus, hepatocytes from zones 3 and 2 of the acinus are differentially responsive to induction by clofibric acid.