Differential expression of transforming growth factor-beta and its receptors in hepatocytes and nonparenchymal cells of rat liver after CCl4 administration

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) is a family of multifunctional proteins that regulate hepatocyte proliferation, and biosynthesis of the extracellular matrix. In this study we examined whether modulation of TGF-beta receptor expression contributes to the liver diseases. METHODS: The mRNA expression of TGF-beta1, TGF-beta type I receptor (TGFbetaRI), TGF-beta type II receptor (TGFbetaRII) and TGF-beta type III receptor (TGFbetaRIII) in rat livers injured by CCl4 administration was studied by Northern blotting. The mRNA expression patterns were confirmed by in situ hybridization. RESULT: The peak of TGF-beta1 mRNA expression was observed 48 h after acute intoxication with CCl4 in nonparenchymal cells. However, the levels of TGFbetaRI and TGFbetaRII mRNA expression decreased from 24 h to 48 h and from 12 h to 48 h, respectively, and returned to the normal level by 72 h. TGFbetaRII mRNA expression was depressed more and for longer than that of TGFbetaRI mRNA. Analysis in separated hepatocytes and nonparenchymal cells from the injured livers indicated that the mRNA changes occurred in hepatocytes. Nonparenchymal cells expressed TGFbetaRI and TGFbetaRII mRNAs at constant levels during liver regeneration. TGFbetaRIII mRNA, which also decreased after 12 h, was not apparent in hepatocytes but only in nonparenchymal cells. CONCLUSIONS: These observations suggest that: (i) whenever TGF-beta1 is increased in CCl4-treated livers, it may induce liver fibrogenesis via nonparenchymal cells; (ii) the mitoinhibitory effect of TGF-beta1 on hepatocytes is transiently relieved by down-regulation of TGF-beta receptors for 72 h post-damage; and (iii) the resistance to TGF-beta growth inhibition between 24 to 48 h may be predominantly due to down-regulation of the expression of TGFbetaRII.     

Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells

Hepatic cells isolated from an adult rat liver, consisting of small hepatocytes (SHs), mature hepatocytes (MHs), liver epithelial cells (LECs), Kupffer cells, sinusoidal endothelial cells, and stellate cells, were cultured in a medium supplemented with 10% fetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL epidermal growth factor, and 1% dimethyl sulfoxide. The SHs rapidly proliferated and formed a colony. About 10% of cytokeratin 8 (CK8)-positive cells formed SH colonies. All SHs at day 10 immunocytochemically showed positivity for albumin, transferrin, CK8, and CK18, which are markers for hepatocytes. In contrast, alpha-fetoprotein (AFP)-, CK14-, OC2-, and glutathione S-transferase placental type (GST-P)-positive cells, which are thought to be markers for hepatic immature cells, were rarely observed. At day 20 some cells in the colonies were positive for AFP, CK7, CK19, and GST-P. LECs and stellate cells proliferated and surrounded the colonies. About 2 weeks after plating, piled up cells were often observed on the SH colonies. In those colonies LECs and stellate cells invaded under the colonies. The invasion of the cells and gradual deposits of extracellular matrix (ECM) such as type I collagen, type IV collagen, and laminin induced alteration of the shape of the SHs from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin, connexin 32 (Cx32), and tryptophan 2,3-dioxygenase (TO) messenger RNAs increased. In addition, overlapping nonparenchymal cells (NPCs) on the piled up cells induced the formation of duct- or cyst-like structures consisting of MHs. In the present experiment we showed that SHs could differentiate to MHs by interacting with NPCs and ECM. Thus, SHs may be "committed progenitor cells" that can further differentiate into MHs.     

Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis

Vascular endothelial growth factor (VEGF) can induce proliferation of sinusoidal endothelial cells. Its mRNA expression was increased in proliferating rat hepatocytes in primary culture. To clarify a role of VEGF in liver after necrosis, expressions of VEGF and its receptors were measured in the liver or liver cells isolated from rats after carbon tetrachloride intoxication. Hepatic VEGF mRNA expression increased later than 24 h after the intoxication and became prominent at 168 h when liver necrosis disappeared, while hepatic mRNA expressions of its receptors increased between 24 and 72 h. VEGF mRNA expression was increased in Kupffer cells, hepatic macrophages and stellate cells isolated from rats between 24 and 72 h after the intoxication and in hepatocytes at 168 h compared to those cells from normal rats. Immunohistochemical VEGF stains were comparable to such results. Vascular endothelial cells existed abundantly in the necrotic areas, and sinusoidal endothelial cells appeared following disappearance of the necrotic areas. VEGF mRNA expression in hepatocytes isolated from 70% resected liver was increased at 12 h after the operation and became marked between 72 and 168 h. Similar increase of hepatic VEGF expression was immunohistochemically seen. In conclusion, VEGF derives from nonparenchymal as well as parenchymal cells in rat liver after necrosis. The former might contribute to vascular endothelial cell proliferation and the latter to sinusoidal endothelial cell regeneration.     

Malarial immunodepression in vitro: adherent spleen cells are functionally defective as accessory cells in the response to horse erythrocytes

The basis for the depressed response of malarial infected mice to horse red blood cells (HRBC) has been studied in vitro. Results presented show that the adherent spleen cells from infected mice (a) are defective in their ability to allow nonadherent spleen cells of both normal and infected mice to respond to HRBC whereas a response does occur with adherent spleen cells from normal mice (b) do not suppress the response of unfractionated spleen cells from normal mice to HRBC (c) contain phagocytic cells as measured by the uptake of neutral red in numbers which are of the same order of magnitude as in adherent spleen cells from normal mice, but which are unable to take up HRBC. We conclude that a splenic adherent cell, probably the macrophage is functionally defective as an accessory cell in the response to HRBC of mice infected with Plasmodium berghei yoelii.     

Inhalation toxicokinetics of butoxyethanol and its metabolite butoxyacetic acid in the male Sprague-Dawley rat

A total of 16 male Sprague-Dawley rats were continuously exposed to 20 ppm or 100 ppm butoxyethanol (BE) vapor for 1, 2, 3, 4, 6, 8, 10, or 12 days. Urine was collected in 24-h intervals and stored at -70 degrees C. At the end of the exposure the animals were euthanized by decapitation and tissue samples of blood, muscle, liver and were rapidly collected and frozen to -70 degrees C. The samples were later derivatized and analyzed for BE and its major metabolite butoxyacetic acid (BAA) by electron capture gas chromatography. BE and BAA were rapidly distributed to the tissues examined. The concentration of BE in blood was slightly higher, and that of BAA markedly higher than in other tissues, indicating weak (BE) and pronounced (BAA) blood protein binding, respectively. BE was efficiently metabolized and the blood clearance averaged 2.6 l/h per kg, corresponding to a hepatic extraction ratio of about 0.75. The renal clearance of BAA (average 0.53 l/h per kg) corresponded to approximately 15% of the renal blood flow. The kinetics of BE and BAA were linear up to 100 ppm. There were no clear indications of changes in the toxicokinetics, such as metabolic induction or inhibition of metabolism or excretion, during the course of the exposure. The recovery of BAA in urine was 64% of the calculated inhaled amount of BE, on an equimolar basis.     

Conditioned medium from activated spleen cells supports the survival of rat retinal cells in vitro

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 microM nifedipine. However, the use of an intracellular calcium chelator (15.0 microM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions.     

Enhanced accumulation of ribavirin and its metabolites in liver versus erythrocytes in mice administered with the liver targeted drug

OBJECTIVE: Determine the frequency and relationship between ischemic heart disease (IHD) and serum cholesterol levels (SCL) in non insulin dependent diabetes mellitus (NIDDM) of the primary medical care level. MATERIAL AND METHODS: A total of 411 patients from the first medical care level were studied. The sociodemographic profile, SCL and glycemia were determined and conventional ECG was taken. The ST uneveness, ischemic T or pathological Q waves in two or more tappings was considered as IHD. Patients with history of IHD were not included. RESULTS: The male:female ratio was 1.5:1. Mean SCL was 225 mg/dl (in females 240.8 +/- 56 mg/dl and 220.7 +/- 50.7 in males). In 90 patients we identified IHD (22%), with male predominance (0.85:1, F:M). In the stratified statistical analysis the SCL > or = 200 mg/dl and IHD were significantly associated. The frequency of IHD by SCL levels of 200-239 mg/dl was 24.6% (OR 2.04; CI 95% 1.03-4.07, p = 0.04) and 24.2% (OR 1.99; CI 95% 1.02-3.96, p = 0.04) for SCL of 240-300 mg/dl; in patients with SCL > 300 mg/dl, an increase of IHD to 38.7% was observed (OR 3.95; CI 95% 1.52-10.30, p = 0.002). CONCLUSIONS: The hypercholesterolemia was one of the most important cardiovascular risk factors in NIDDM, in which SCL > or = 200 mg/dl must be considered strongly associated to IHD.     

Effect of perchloroethylene and its metabolites on intercellular communication in clone 9 rat liver cells

Gap junction intercellular communication (IC) is thought to be important in chemical carcinogenesis as abnormalities in IC have been found in cancer cells. Perchloroethylene (PERC) is metabolized in rodent liver to dichloroacetic acid (DCA) and trichloroacetic acid (TCA), which are rodent liver carcinogens. Chloral hydrate (CH) and trichloroethanol (TCEth) are kidney metabolites. We used Lucifer yellow scrape-load dye transfer as a measure of IC to look at the effect of PERC, DCA, TCA, CH, and TCEth on Clone 9 cell cultures (normal rat liver cells). Four independent experiments were performed for each chemical using exposure times of 1, 4, 6, 24, 48, and 168 h. Concentrations for each chemical varied and were based on preliminary data on effect and cytotoxicity. To compare the relative effectiveness of each chemical to cause biological change, we identified the lowest concentration and shortest time to significantly reduce dye transfer. DCA caused a significant change at 10 mM at 6 h; TCA, 1 mM at 1 h; CH and TCEth, 1 mM at 24 h; and PERC, 0.01 mM at 48 h. Over a 24-h treatment period, the relative efficiencies, as defined by the concentration needed to produce 50% reduction in IC, were PERC (0.3 mM) > TCA (3.8 mM) > TCEth (6.6 mM) = CH (7.0 mM) > DCA (41 mM). Time-course data indicated that PERC, DCA, and TCA produced reduction in IC in a similar fashion, but 5 mM CH or TCEth exhibited variances from these results and may indicate specific cell responses to these chemicals. The mechanism(s) responsible for inhibition of IC by these structurally related chemicals needs to be established.     

State of rat liver and spleen DNA after space flight on the "Cosmos-605" satellite

The effect of space flight on the content of nucleic acids and the structure of DNA in the rat liver and spleen was studied. The 22-day exposure did not influence the DNA content in tissues, whereas the RNA content decreased by 14% and 19% in the liver and spleen, respectively. The flight did not induce changes like one-stranded or paired breakages in the DNA structure. Studies of the liver showed a minor trend towards a fall of elastic viscosity of the DNA supermolecular structure (difference between the flight and control rats was statistically insignificant). The above changes in the state of nucleic acids in the liver and spleen were transient.     

Lead in the blood and protoporphyrin levels in erythrocytes in glass workers exposed to lead]

The craniofacial cephalometric dimensions, angles and dimensional ratios of five Finnish individuals with complete testicular feminization (CTF) were compared with their first-degree relatives and population female and male controls. The linear and angular measurements were made from standardized lateral cephalograms of patients and normal population controls from the 'Kvantti Study' series. The women with CTF tended to have cranial base and maxillary complex dimensions between those of the normal control females and males. Their mandibular corpus was found to be longer than in normal control females, while their ramus was shorter compared with that of normal males. They also showed a smaller sagittal length ratio of the maxilla to the mandible, a smaller ANB angle and a more acute gonial angle than in both normal control females and males. Comparison of the women CTF with their first-degree female relatives showed basically the same trends as when comparing them with normal female controls. As the phenotype in these females with CTF is due to insensitivity to, or lack of androgens, it is suggested that the presence of the Y chromosome in these females leads to craniofacial dimensions between those of normal females and males which influences the growth of the mandibular corpus. This follows the same general metric pattern that is observed in many of their adult head and body dimensions as well as in their dental arches.     

Expression and activity of urokinase and its receptor in endothelial and pulmonary epithelial cells exposed to asbestos

Antisera against peptides from the extreme N- and C-terminal regions of the VDR were evaluated. The N-terminal antiserum Ab192 functioned as a general-purpose antibody, being able to supershift the rhVDR in heterodimeric or homodimeric binding complexes in the EMSA, and detect both recombinant and native forms of the receptor on Western blots. The C-terminal antiserum, Ab195, also identified the receptor on Western blots, but in contrast, it displayed differential sensitivity to the conditions employed in the EMSA. In the presence of 1,25(OH)2D3, rhVDR, rhRXR alpha, and nonspecific DNA, Ab195 produced a weak supershift of the heterodimer complex in the EMSA. Significantly, omission of hormone from the binding buffer resulted in a complete shift of the bound complex with the antiserum. A complete supershift was also observed if only the nonspecific DNA was removed. Together these results indicate antiserum sensitivity to the ligand status in the rhVDR C-terminus as part of a DNA-bound heterodimer complex. Inclusion of 9-cis RA resulted in a modest increase in the amount of shifted product relative to 1,25(OH)2D3 alone. Finally, Ab195 completely supershifted the rhVDR homodimer binding complex under all tested conditions, including those analogous to where it was largely ineffective in shifting the heterodimer. Thus, Ab195 is sensitive to the ligand binding status of the VDR, discriminates heterodimer and homodimer binding interactions, and should provide an additional tool for exploring conformational changes induced in the receptor.     

The effect of indolinic and quinolinic nitroxide radicals on trout erythrocytes exposed to oxidative stress

We have undertaken a clinical and molecular study of 25 females with deletions of the short arm of the X chromosome. We have determined the deletion breakpoints, the parental origin and the activation status of the deleted X chromosomes. Genotype-phenotype correlations suggest that the presence of a single copy of the DFFRX gene, previously postulated as a gene involved in the ovarian failure seen in Turner syndrome, may be compatible with normal ovarian function, and that there may be a gene for Turner-like features located in distal Xp22.3.     

Comparison between the effects of D-mannoheptulose and its hexaacetate ester upon D-glucose metabolism in rat erythrocytes

At a 2.0 mM concentration, D-mannoheptulose hexaacetate, but not unesterified D-mannoheptulose, inhibited the generation of radioactive acidic metabolites by rat erythrocytes exposed to D-[U-14C]glucose (8.3 mM). It is proposed that D-mannoheptulose hexaacetate represents a valuable tool to interfere with the phosphorylation of D-glucose in cell types otherwise resistant to the heptose.     

Bioactivation of estrone and its catechol metabolites to quinoid-glutathione conjugates in rat liver microsomes

Although the carcinogenic effects of estrogens have been mainly attributed to hormonal properties, there is interest in estrogens acting as chemical carcinogens by binding to cellular macromolecules. In the present study, we explored factors which influence the rate of P450-catalyzed formation of the o-quinones (3,5-cyclohexadiene-1,2-diones) from 2-hydroxyestrone (2-OHE) and 4-hydroxyestrone (4-OHE) as well as from estrone in rat liver microsomes. The initially formed o-quinones were trapped as their GSH conjugates which were separated and characterized by HPLC with electrospray-MS detection. Two mono-GSH conjugates were observed from the 2-OHE-o-quinone as well as a conjugate where GSH had added twice to the molecule producing a di-GSH conjugate. 4-OHE-o-quinone gave only one mono-GSH adduct as well as a di-GSH adduct. Both 2-OHE and 4-OHE were excellent substrates for P450, generating o-quinone GSH adducts at 94 and 40 times, respectively, the rate of estrone. 2-OHE but not 4-OHE saturated P450 at unusually low concentrations (0.2 nmol of P450/mL) perhaps due to differences in the stability of the o-quinones formed in the active site of the enzyme. Preliminary data suggest that the o-quinones of both 2-OHE and 4-OHE could isomerize to quinone methides (4-alkyl-2,5-cyclohexadien-1-ones, QMs). The o-quinones of the catechol estrogens were incubated at 37 degrees C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combined with GSH. From the pseudo-first-order rate of disappearance of the o-quinone GSH adducts, the half-lives of the o-quinones were determined. The o-quinone from 2-OHE has a half-life of 42 +/- 3 s at 37 degrees C (pH 7.4), and the o-quinone from 4-OHE has a half-life of 12.2 +/- 0.4 min under identical conditions. The o-quinones of the AB ring analogs of the catechol estrogens (3,4-dihydroxy-5,6,7,8-tetrahydronaphthalene and 1,2-dihydroxy-5,6,7,8-tetrahydronaphthalene) isomerize to QMs, suggesting that a similar reaction pathway could occur with the o-quinones from catechol estrogens. In support of this, oxidation of 4-OHE and quenching with GSH after 70 min produced 9-dehydro-4-hydroxyestrone (3-hydroxy-1,3,5-(10),9(11)-estratetraen-17-one), a product which could result from either the QM hydrolysis product or the QM--glutathione conjugate, both of which could eliminate to give the conjugated alkene of 4-OHE. The implications of the o-quinone/QM pathway to the in vivo effects of catechol estrogens are not known; however, given the direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.     

Analysis of cell cycle disruptions in cultures of rat pleural mesothelial cells exposed to asbestos fibers

The control of DNA integrity in mammalian cells is important to maintain the cell homeostasis and prevent neoplastic transformation. Control of cell division and cell death permits repair or elimination of damaged cells. Since asbestos fibers can produce DNA damage, chromosome alterations and apoptosis in several sorts of cells, including mesothelial cells, it was interesting to investigate cell cycle disturbances in rat pleural mesothelial cells (RPMC) treated with asbestos fibers. Cell cycle analyses were performed in RPMC exposed to crocidolite (10 and 20 microg/cm2) and chrysotile (5 and 10 microg/cm2) for different times (4 to 48 h). Both fiber types entailed a G2/M accumulation in agreement with a delay in the mitosis course. Chrysotile fibers produced a G0/G1 accumulation associated with a time-dependent p53 and p21 expression. Crocidolite exposure resulted in a delay in the G1/S transition paralleling a low rate of p53 expression. These results are in agreement with a DNA damaging potential of asbestos fibers since similar results were found following RPMC exposure to gamma rays. In asbestos-treated RPMC, a low rate of apoptosis was found suggesting that RPMC may follow a DNA repair pathway that could contribute to the formation of DNA lesions. In addition, the cell cycle disturbances at the G2/M checkpoint suggest that genetically altered cells have progressed through the cycle and support the already published findings on the ability of asbestos fibers to impair cell division.     

Antibody-coated sheep erythrocytes suppress the ability of polyclonal B-cell activators to induce plaque-forming cells against sheep erythrocytes

The primary immune response to untreated sheep erythrocytes (SRBC) in vitro was suppressed by the addition of antibody-coated SRBC. A mixture of SRBC and antibody-coated SRBC also suppressed the induction of anti-SRBC plaque-forming cells by the polyclonal B cell activators lipopolysaccharide, purified protein derivative of tuberculin, and native dextran. Injection of a mixture of SRBC and antibody-coated SRBC into mice led to an increased response to SRBC. It seems plausible from the in vitro findings that the Fc part of antibodies complexed to an antigen can exert a negative signal on antigen-specific B cells that cannot be overcome by positive signals delivered by polyclonal B cell activators.     

Effect of cytokine-specific antisense oligonucleotides on the immunoglobulin production by rat spleen cells in vitro

We examined in this study the regulation of immunoglobulin (IgM, IgG, IgE) production by spleen cells from N brasiliensis infected rats following addition of antisense oligodeoxynucleotides (ODNs). The ODNs were selected near the AUG initiation codon of mRNA specific for interleukin 4 (IL-4) or interleukin 2 (IL-2). Results show that addition of antisense to IL-4 inhibited IgE production, while the production of IgG and IgM increased. The use of sense IL-4 sequence did not affect immunoglobulin production. In contrast, the use of antisense IL-2 ODN induced an enhancement of IgE as well as of IgM and IgG responses. Both the Ig secretion in culture supernatants and the number of Ig secreting cells, as detected by an Elispot assay, were influenced by the presence of antisense IL-4 ODNs. These results clearly show the involvement of IL-2 and IL-4 in the regulation of isotype selection during antibody synthesis and that IL-2 and IL-4 do operate differently on IgE production. They also argue that antisense strategy represents a useful tool for the antibody regulation.     

Metallothionein content increased in the liver of mice exposed to magnetic fields

OBJECTIVES: The aim of this investigation was to evaluate the significance of selected surface texture parameters used to describe and quantify the effect of tooth brushing with various "tooth whitening" dentifrices on a resin composite surface in vitro. METHODS: Specimens of a microfil resin composite were brushed with selected dentifrices. Surface texture profiles were acquired and analyzed both pre- and post-brushing using a contact diamond stylus. The selected parameters chosen to describe the surface texture were Ra, Rz, Rpm and the Rpm:Rz ratio. Differences between toothpastes were assessed using an ANOVA and a multiple comparisons test, the Student Newman-Keuls procedure. P and t values were calculated to determine if any of the surface roughness parameters were significantly changed by brushing. RESULTS: The results indicate that there were significant changes in the surface texture of the resin composite following tooth brushing with the selected dentifrices. For example, the use of Clinomyn significantly increased the surface roughness of the resin composite, as measured by the Rz parameter, from 2.19 +/- 1.67 microns to 10.02 +/- 2.57 microns (p < 0.05). In addition, the surface texture parameters chosen to describe the properties of the surface should reflect a knowledge of profile shape such as Rpm:Rz ratio, and care should be taken if measurements of surface texture of dental restorative materials are to be used as predictors of clinical performance. SIGNIFICANCE: All the toothpastes chosen for this investigation left a surface on the resin composite which may be prone to crack propagation during "vertical barrelling" movements generated during mastication. However, this may be more of a function of the rigidity of the restorative material rather than the surface left after tooth brushing.