Zebrafish developmental screening of the ToxCast™ Phase I chemical library.

Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecology. As part of the Computational Toxicology Research Program of the U.S. EPA, the toxicity of the 309 ToxCast™ Phase I chemicals was assessed using a zebrafish screen for developmental toxicity. All exposures were by immersion from 6-8 h post fertilization (hpf) to 5 days post fertilization (dpf); nominal concentration range of 1 nM-80 µM. On 6 dpf larvae were assessed for death and overt structural defects. Results revealed that the majority (62%) of chemicals were toxic to the developing zebrafish; both toxicity incidence and potency was correlated with chemical class and hydrophobicity (logP); and inter-and intra-plate replicates showed good agreement. The zebrafish embryo screen, by providing an integrated model of the developing vertebrate, compliments the ToxCast assay portfolio and has the potential to provide information relative to overt and organismal toxicity.

Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR.

The recently identified placenta growth factor (PIGF) is a member of the vascular endothelial growth factor (VEGF) family of growth factors. PIGF displays a 53% identity with the platelet-derived growth factor-like region of VEGF. By alternative splicing of RNA, two PIGF isoforms are generated: PIGF131 (PIGF-1) and PIGF152 (PIGF-2). Relative to PIGF131, PIGF152 has a 21-amino acid insertion enriched in basic amino acids. Little is known at the present time about the significance and function of these proteins. To assess their potential role, we cloned the cDNAs coding for both isoforms, expressed them in mammalian cells, and purified to apparent homogeneity the recombinant proteins. Like VEGF, the PIGF isoforms are homodimeric glycoproteins. PIGF131 is a non-heparin binding protein, whereas PIGF152 strongly binds to heparin. We examined the ability of PIGF to bind to soluble VEGF receptors, Flt-1 and Flk-1/KDR, and characterized the binding of PIGF to endothelial cells. While the PIGF proteins bound with high affinity to Flt-1, they failed to bind to Flk-1/KDR. Binding of 125I-PIGF to human endothelial cells revealed two classes of sites, having high and low affinity. The high affinity site is consistent with Flt-1; the identity of the low affinity site remains to be determined. Purified PIGF isoforms had little or no direct mitogenic or permeability-enhancing activity. However, they were able to significantly potentiate the action of low concentrations of VEGF in vitro and, more strikingly, in vivo.

Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms.

The vascular endothelial growth factor (VEGF) family encompasses four polypeptides that result from alternative splicing of mRNA. We have previously demonstrated differences in the secretion pattern of these polypeptides. Stable cell lines expressing VEGFs were established in human embryonic kidney CEN4 cells. VEGF121, the shortest form, was secreted and freely soluble in tissue culture medium. VEGF189 was secreted, but was almost entirely bound to the cell surface or extracellular matrix. VEGF165 displayed an intermediary behavior. Suramin induced the release of VEGF189, permitting its characterization as a more basic protein with higher affinity for heparin than VEGF165 or VEGF121, but with similar endothelial cell mitogenic activity. Heparin, heparan sulfate, and heparinase all induced the release of VEGF165 and VEGF189, suggesting heparin-containing proteoglycans as candidate VEGF-binding sites. Finally, VEGF165 and VEGF189 were released from their bound states by treatment with plasmin. The released 34-kDa dimeric species are active as endothelial cell mitogens and as vascular permeability agents. We conclude that the bioavailability of VEGF may be regulated at the genetic level by alternative splicing that determines whether VEGF will be soluble or incorporated into a biological reservoir and also through proteolysis following plasminogen activation.

The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA.

Vascular endothelial growth factor (VEGF) was recently identified as a secreted, direct-acting mitogen specific for vascular endothelial cells and capable of stimulating angiogenesis in vivo. Molecular cloning revealed multiple forms of VEGF, apparently arising from alternative splicing of its RNA transcript. We have examined various human cDNA libraries by the polymerase chain reaction technique and discovered a fourth molecular form, VEGF206. This form contains a 41-amino acid insertion relative to the most abundant form, VEGF165, and includes the highly basic 24-amino acid insertion found in VEGF189. Southern blot analysis revealed that a single gene encoded these various forms, and nucleic acid sequence analysis of a portion of the VEGF gene revealed an intron/exon structure compatible with alternative splicing of RNA as a mechanism for their generation. Transient transfection of human embryonic kidney 293 cells showed that, like VEGF189, VEGF206 was predominately cell-associated and only very poorly secreted despite the presence of the signal peptide identical to that found in VEGF121 and VEGF165, both of which are efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF; however, endothelial cell mitogenic activity was apparent only with VEGF121 and VEGF165. Thus, alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.

The vascular endothelial growth factor family of polypeptides.

Vascular endothelial growth factor (VEGF) was identified as a heparin-binding polypeptide mitogen with a target cell specificity restricted to vascular endothelial cells. Molecular cloning reveals the existence of four species of VEGF having 121, 165, 189, and 206 amino acids. These have strikingly different secretion patterns, which suggests multiple physiological roles for this family of polypeptides. The two shorter forms are efficiently secreted, while the longer ones are mostly cell-associated. Alternative splicing of mRNA, rather that transcription from different genes, is the mechanism for their generation. In situ hybridization reveals that the VEGF mRNA is widely distributed in most tissues and organs and expressed at particularly high levels in areas of active vascular proliferation, like the ovarian corpus luteum. Ligand autoradiography on rat tissue sections demonstrates that VEGF binding sites are associated with vascular endothelial cells of both fenestrated and non-fenestrated capillaries and with the endothelium of large vessels, while no displaceable binding is evident on non-endothelial cell types. These findings support the hypothesis that VEGF plays a highly specific role in the maintenance and in the induction of growth of vascular endothelial cells.

Acidic fibroblast growth factor (HBGF-1) stimulates DNA synthesis in primary rat hepatocyte cultures.

Acidic fibroblast growth factor (aFGF) stimulated DNA synthesis in primary rat hepatocyte cultures in a dose-dependent manner with maximal effect at 10-50 ng ml-1. This activity was dependent on the presence of heparin at a concentration of 10-50 micrograms.ml-1. Insulin interacted synergistically with aFGF, as it did with epidermal growth factor (EGF). The response to aFGF was only 50% that found with EGF. The disparity was not due to different kinetics of DNA synthesis, since the peak response for both growth factors occurred at 36-72 hr after plating of the hepatocytes. The potential relevance of this novel hepatocyte mitogen to normal and pathological liver growth is discussed.

Ecological toxicology and human health effects of heptachlor.

The chlorinated cyclodiene heptachlor was registered in 1952 as an agricultural and domestic insecticide. By early 1984, registration for all purposes, except subterranean termite control and for limited use in the control of fire ants, had been cancelled. This restriction of use arose primarily from concerns over the environmental persistance and bioaccumulation potential of the organochlorine pesticides. Currently, sale of heptachlor has been voluntarily suspended over questions about its carcinogenic potential, and the absence of safe and effective application methods. As a persistent organochlorine pesticide, heptachlor residues are detected in all components of the environment. In historical use, heptachlor was directly applied to terrestrial systems, while air and water were secondarily contaminated via volatilization and land run-off, respectively. Within each environmental compartment, heptachlor undergoes a variety of metabolic and abiotic transformations. In vivo studies indicate that heptachlor epoxide is the predominant metabolite, formed as a product of the mixed-function oxidase system, while 1-hydroxychlordene is the major soil metabolite. For quantification, heptachlor and its metabolites are extracted from air, soil and sediment, water, or biological materials using various organic solvents and analyzed by gas chromatography or thin-layer chromatography. Residue reports comprise most of the literature concerning the effects of heptachlor on the biota. In many such reports, toxic effects cannot be conclusively attributed to heptachlor exposure. Toxicity to organisms seems more dependent on acute exposure, while the chronic effects of low level exposure to heptachlor are poorly defined. Maximal terrestrial residues coincide with temporal and spatial proximity to application; peak residues in aquatic systems on the other hand, correlate to periods of maximum run-off. The lipophilic nature of both heptachlor and heptachlor epoxide results in the potential for significant bioaccumulation in all lipid-type compartments in the environment. The toxic effects of heptachlor are not specific for any one organ system. The liver and the central nervous system are most significantly affected by heptachlor, although effects can also be seen in the reproductive, hematopoietic, immune, and renal systems. An important consideration is the relation of relevant environmental exposure levels to toxicity. The concentrations necessary to elicit results in laboratory experiments do not translate directly to the same results upon environmental exposure, nor do experimental laboratory animal models absolutely equate with native-state organisms or with humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered responses of regenerating hepatocytes to norepinephrine and transforming growth factor type beta.

Norepinephrine (NE), acting through the alpha 1-adrenergic receptor, modules the response of rat hepatocytes in primary culture to transforming growth factor type beta 1 (TGF beta) by increasing the amount of TGF beta required for a given degree of inhibition of epidermal growth factor (EGF)-induced DNA synthesis (Houck et al., J. Cell. Physiol. 135:551-555, 1988). This effect was also found in hepatocytes isolated from regenerating livers but was greatly magnified in cells isolated between 12 and 18 hr after two-thirds partial hepatectomy (PHX). During this period of enhanced sensitivity, NE was equally potent in terms of dose but more efficacious in the regenerating hepatocytes. As it did in control hepatocytes (Cruise et al., Science 227:749-751, 1985), the alpha 1-adrenergic receptor mediated the activity of NE in regenerating hepatocytes. Vasopressin (VP) and angiotensin-II (AG) also antagonized the effect of TGF beta and showed increased activity in regenerating hepatocytes but at only 50% or less of the maximal effect reached by NE. Regenerating hepatocytes isolated 24-72 hr after PHX exhibited decreased sensitivity to inhibition by TGF beta, with a nadir in 48-hr-regenerating cells. These findings suggest that NE may be involved in triggering the early phase of DNA synthesis during liver regeneration, with the subsequent acquisition of innate resistance to TGF beta responsible for continued proliferation at a time when TGF beta mRNA is known to be increasing in the liver (Braun et al., Proc. Natl. Acad. Sci. USA 85:1539-1543, 1988). EGF induced increased DNA and protein synthesis in cultures of control hepatocytes; TGF beta inhibited the EGF-induced DNA synthesis but had no effect on protein synthesis. This may be relevant to the latter stages of liver regeneration, when high levels of TGF beta mRNA are detected in liver and cellular hypertrophy predominates over hyperplasia.

Introduction of a Ha-ras oncogene into rat liver epithelial cells and parenchymal hepatocytes confers resistance to the growth inhibitory effects of TGF-beta.

Growth of rat liver epithelial cells (RLEC) and primary cultures of parenchymal hepatocytes is potently inhibited by TGF-beta. Transfection of a mutated Ha-ras oncogene, but not a human c-myc oncogene, into RLEC resulted in cell lines resistant to growth inhibition by TGF-beta under anchorage-dependent conditions. Infection of primary rat hepatocyte cultures with v-Ha-ras yielded a cell line likewise insensitive to inhibition by TGF-beta. Binding of [125I]TGF-beta to Ha-ras-transfected RLEC was reduced relative to control or c-myc-transfected cells. These data suggest that activation of a Ha-ras oncogene in epithelial cells may result in escape from negative growth control and hence be a critical step during carcinogenesis. However, although Ha-ras induced resistance to growth inhibition by TGF-beta under anchorage-dependent conditions, TGF-beta inhibited the spontaneous growth in soft agar of all cell lines containing the Ha-ras oncogene. This may reflect an alteration in regulation of extracellular matrix proteins and related enzymes responsible for anchorage-independent growth.

Norepinephrine modulates the growth-inhibitory effect of transforming growth factor-beta in primary rat hepatocyte cultures.

TGF-beta is a potent inhibitor of EGF-induced DNA synthesis in primary rat hepatocyte cultures. Norepinephrine (NE) was shown to modulate this inhibition of DNA synthesis. It produced a five-fold increase, from 2.8 pM to 14.4 pM, in the ID50 for TGF-beta. The effect was dose-dependent and was significant at concentrations of 10(-6)M NE and greater. The modulation by NE was mediated by the alpha 1-adrenergic receptor as shown by the ability of the alpha 1 antagonist prazosin to block the activity. This effect might be important during liver regeneration in allowing escape of hepatocytes from negative growth control exerted by TGF-beta.

Early events in the regulation of hepatocyte DNA synthesis: the role of alpha-adrenergic stimulation.

The role of adrenergic agents in DNA synthesis was investigated in two models of stimulated hepatocyte growth: in vitro primary serum-free cultures of adult parenchymal hepatocytes, and in vivo liver regeneration after two-thirds partial hepatectomy. In both systems the alpha 1-adrenergic receptor appeared to be involved in mediating stimulatory effects. In primary hepatocyte cultures norepinephrine acted via this receptor to enhance the DNA synthesis stimulated by epidermal growth factor (EGF), and heterologously downregulated EGF receptors. In liver regeneration the administration of an alpha 1 blocking agent interfered with the first wave of regenerative DNA synthesis, and this effect was preceded by an elevation in EGF receptor number. Measurements of plasma catcholamines demonstrated that elevated levels of norepinephrine and epinephrine were in circulation within 2 h after partial hepatectomy. Surgical hepatic sympathectomy also interfered with early liver regeneration, suggesting that locally delivered adrenergic agents are important to initiation of DNA synthesis. These data suggest that stimulation at the alpha 1-adrenergic receptor is among the early signals for liver regeneration and that heterologous regulation of EGF receptors, similar to that observed in vitro, may be a part of the regenerative response.

Differential effect of growth factors on growth stimulation and phenotypic stability of glutamine-synthetase-positive and -negative hepatocytes in primary culture.

In rat liver parenchyma, two subpopulations of hepatocytes can be distinguished by the absence or presence of the marker enzyme, glutamine synthetase (GS). Hepatocytes in the perivenous zone immediately adjacent to the hepatic venules in the liver acinus are positive for GS. Using autoradiography in combination with immunocytochemistry, the response of these two hepatocyte populations (GS positive and GS negative) to a variety of growth factors (defined compounds or complex stimuli) was investigated in vitro. Irrespective of the individual growth-promoting activity (which varied considerably), all stimuli led to much higher labeling indices in GS-negative cells as compared to GS-positive cells. In GS-negative cells, the strongest effect was exerted by serum obtained from partially hepatectomized rats (labeling index, 67%) and the conditioned media of JM1 and JM2 hepatoma cells (63%-82%), followed by a combination of insulin and either norepinephrine (46%) or epidermal growth factor (EGF; 42%). In contrast, serum had the weakest influence on GS-positive cells (0.3%), while the other potent stimuli enhanced the labeling index of these cells by between 6% and 15% within 48 h. The percentage of labeled nuclei was higher in mononucleated than in binucleated GS-positive hepatocytes. The time course of thymidine incorporation was also different for the two subpopulations. Under all growth-promoting conditions, the stimulation of GS-negative cells peaked between 72 and 96 h, while it increased continuously in GS-positive cells for at least 120 h, particularly in the case of serum. In proliferating cultures, both the absolute and the relative number of GS-positive hepatocytes decreased, while no such effect was found in various nonproliferating control cultures maintained at low and high cell density. Similar results were found for GS activity. In contrast, the hormonal induction of tyrosine aminotransferase (TAT) was not affected. It is suggested that these differences in the growth response of GS-positive and -negative cells contribute to the acinar gradient in hepatocyte proliferation that occurs during liver regeneration. Furthermore, the striking phenotypic instability of GS-positive cells that have undergone DNA synthesis and mitosis supports the hypothesis that cellular reprogramming depends on passage through the cell cycle.

Induction of DNA synthesis in cultured rat hepatocytes through stimulation of alpha 1 adrenoreceptor by norepinephrine.

Addition of norepinephrine to primary cultures of adult rat hepatocytes stimulates the incorporation of [3H]thymidine in a dose-dependent manner. This effect has been observed in serum-free medium containing epidermal growth factor and insulin. Stimulation of DNA synthesis by norepinephrine was strongly antagonized by the alpha 1-adrenergic antagonist prazosin but not by an alpha 2 antagonist or by a beta-adrenergic blocker. The beta agonist isoproterenol did not stimulate significant DNA synthesis. These results indicate that catecholamines interact with the alpha 1 adrenoreceptor to stimulate DNA synthesis in hepatocytes. Since alpha 1 receptors are present in most cells, this receptor may be important in cell growth regulation.

Proline is required for the stimulation of DNA synthesis in hepatocyte cultures by EGF.

Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis in rat parenchymal hepatocytes both in vivo in vitro. We report here that this response in vitro is dependent on the amino acids present in the media. Of all the amino acids, proline has the strongest effect. The response to EGF is absent without proline and none of the other amino acids can substitute for it. Added proline (1 mM) to the media caused the labeling index to increase from 11% to 55% in the presence of 50 ng/ml EGF and insulin. In the presence of proline, small additional increases of the EGF effect on DNA synthesis were stimulated by phenylalanine and tyrosine.

Control of hepatocyte replication by two serum factors.

Serum proteins from hepatectomized or control rats were separated by gel permeation chromatography and assayed for stimulation of hepatocyte proliferation in primary cultures of hepatocytes. Two peaks of activity were seen in the areas of large (greater than 120,000) and small (less than 3,000) molecular weight. These activities are different from insulin, epidermal growth factor, or vasopressin and are empirically termed hepatopoietin A and B, respectively. The two activities interact in a synergistic manner to stimulate hepatocyte proliferation at rates comparable to that of the whole serum.