Protective action of dehydroascorbic acid on the Ah receptor-dependent and receptor-independent induction of lipid peroxidation in adipose tissue of male guinea pig caused by TCDD administration

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on lipid peroxidation, ³H-Me-glucose (³H-Me-glu), and ¹⁴C-dehydroascorbic acid (¹⁴C-DHA) uptakes were studied in adipose tissue of male guinea pig. Under in vitro test conditions, using isolated adipose tissue in a culture medium (explant culture), TCDD reduced the uptake of ³H-Me-glu and ¹⁴C-DHA in a dose- and time-dependent fashion. The IC₅₀ values of TCDD's action were 0.04 and 2 nM on ¹⁴C-DHA and ³H-Me-glu uptakes, respectively. TCDD (10 nM) also suppressed glucose transporting activity within 15 minutes in explant-cultured adipocytes. Cytochalasin B (CB) and nonlabeled D-glucose inhibited ¹⁴C-DHA uptake also in a dose-dependent manner. In addition, TCDD was found to induce lipid peroxidation in ex-plant-cultured adipose tissue. This effect of TCDD was similar to that of a typical lipid peroxidation inducer, CCl⁴, and it was dose and time dependent. TCDD caused a statistically significant rise in lipid peroxidation at a concentration as low as 0.1 nM after 60 minutes of treatment in explant culture. Unexpectedly, the Ah receptor partial antagonists, 4,7-phenanthroline and α-naphthoflavone, did not fully antagonize TCDD-induced lipid peroxidation in explant-cultured adipocytes. In vivo treatment of TCDD also induced lipid peroxidation. Among seven organs of male guinea pig tested, the levels of lipid peroxidation in adipose tissue and in liver increased at 1 and 40 days following a single i.p. dose of TCDD (1 μg/kg). The results of an in vivo time-course study indicated that such an effect of TCDD was most pronounced after 40 days of treatment. Finally, we have tested the protective role of some antioxidants on TCDD-induced lipid peroxidation under explant-culture conditions. The results indicated that DHA, but not ascorbic acid, could completely abolish TCDD-induced lipid peroxidation. The protective effect of DHA on TCDD-induced lipid peroxidation was stronger than that of α-tocopherol and uric acid, and this effect was blocked by CB. We conclude from these studies that TCDD acts in this guinea pig tissue through two different routes: one is the Ah receptor-dependent route causing the reduction of the level of glucose transporters and subsequent decrease of cellular uptake of DHA and the other, the Ah receptor-independent route causing the overall lipid peroxidation. Nevertheless, it appears likely that both events are antagonized by DHA. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 269–278, 1997.     

Ah receptor involvement in mediation of pyruvate carboxylase levels and activity in mice given 2,3,7,8-tetrachlorodibenzo-p-dioxin

The arylhydrocarbon receptor (AhR) plays a central role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in animals. The investigations described here provide evidence that support a role for the AhR in TCDD-mediated pyruvate carboxylase (PC) level/activity reductions in mice. Pyruvate carboxylase plays a pivotal role in gluconeogenesis and in supplying carbon units for the citric acid cycle. Delivered ip in a corn oil carrier, TCDD suppresses PC activity/amount at doses as low as 1 μg/kg in responsive C57BL/6J(Ah^b/b) mice. Corn oil alone injected ip into mice at 4 mL/kg appears to be an inducer that increases the amount and activity of PC. However, TCDD suppresses this induction. In the Ah^b/b mouse, PC levels and activity are reduced to 10% of control values at a dose of 75 μg/kg. A time-course experiment shows that the PC reductions are apparent within 16 hours post-TCDD exposure. Here we report investigations on the PC/TCDD response using a congenic C57BL/6J(Ah^d/d) mouse strain having an AhR with a low affinity for TCDD. If the PC/TCDD response is AhR mediated, the congenic mouse strain (Ah^d/d) would require much higher doses of TCDD to suppress PC. In the Ah^d/d mice, we observe that an approximately 60-fold increase in TCDD dose is necessary to produce a PC/TCDD effect. We also find that in Ah^d/d mice, corn oil does not induce an increase in PC activity/amounts, as reported for Ah^b/b mice.     

Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity

We have suggested previously that the amino-terminal 8 kilodaltons of pp60^src may serve as a structural hydrophobic domain through which pp60^src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60^src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, in contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60^src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60^src, and suggest that the amino terminus of pp60^src may have a critical role in the promotion of in vivo tumorigenicity.     

Characterization of a normal avian cell protein related to the avian sarcoma virus transforming gene product.

In this paper, we identify and characterize both structurally and functionally a protein from normal uninfected avian cells that is antigenically related to the pp60^src viral protein responsible for transformation by ASV. This protein was detected by immunoprecipitation of radiolabeled normal cell extracts with serum derived from marmosets bearing ASV-induced tumors. The normal avian cell protein, which has been detected in each of the four avian species tested (chicken, duck, quail and pheasant) is a phosphoprotein of 60,000 daltons. This protein is not related to any of the ASV structural proteins; however, its immunoprecipitation is prevented by preadsorption of the antiserum with cell extracts specifically containing pp60^src. Peptide analyses by partial proteolysis using chymotrypsin resulted in a map of the normal cell protein that was very similar to that of pp60^src. When Staphylococcus aureus V8 protease was used, however, one of the major cleavage products of the normal cell protein exhibited an altered migration with respect to the corresponding pp60^src product. Tryptic phosphopeptide analyses demonstrated that phosphorylation of the normal cell protein was also different from that seen in pp60^src. The expression of the normal cell protein did not seem to be affected by cellular growth conditions, maintaining a constant level which was approximately 30–50 fold lower than that of pp60^src in infected cells. The normal cell protein appeared to be functionally dissimilar to pp60^src lacking detectable protein kinase activity in the currently available assay system.     

The specific interaction of the Rous sarcoma virus transforming protein, pp60src, with two cellular proteins

Sera from rabbits bearing tumors induced by Rous sarcoma virus (RSV) were previously found to contain antibody to the RSV transforming protein, pp60^src. Two additional transformation-specific phosphoproteins from RSV-transformed avian cells are immunoprecipitated with these sera. These proteins, having molecular weights of 90,000 (pp90) and 50,000 (pp50), are not precipitated from uninfected or transformation-defective virus-infected cells and are not related to any RSV structural proteins. Neither pp50 nor pp90 shares any partial or complete proteolytic cleavage peptides with pp60^src, suggesting that pp90 and pp50 do not represent either a precursor or a cleavage product of pp60^src. Sedimentation analysis of RSV-transformed cell lysates on glycerol gradients revealed that the RSV pp60^src protein is present as two forms, one of which represents the majority (95%) of pp60^src and sediments as a monomer, 60,000 molecular weight protein and the other of which sediments with pp90 and pp50 as an apparent 200,000 molecular weight complex. Lysates from cells transformed by viruses containing a temperature-sensitive defect in the src gene contain a greater percentage of pp60^src associated with pp90 and pp50 under both permissive (35°C) and nonpermissive (41°C) conditions compared to wild-type virus-infected cell lysates. Phosphoserine and phosphotyrosine were found associated with pp60^src molecules that sedimented as a monomer, whereas pp60^src molecules that are complexed with pp90 and pp50 contain phosphoserine and greatly reduced amounts of phosphotyrosine. Only the monomer form of pp60^src is capable of phosphorylating IgG in the immune complex phosphotransferase reaction. Normal uninfected chicken cells contain a protein that shares identical partial proteolytic cleavage peptides with the pp90 protein immunoprecipitated from RSV-transformed cells. This pp90 protein is one of the major cytoplasmic proteins in uninfected cells. Antibody directed against pp90 also immunoprecipitates pp60^src and pp50 from lysates of RSV-transformed chicken cells.     

Cytoskeletal association of pp60 : The transforming protein of the rous sarcoma virus

Immunoferritin labelling methods have been employed to examine the distribution of the Rous Sarcoma virus (RSV)-transforming protein pp60^src in the detergent-resistant cytoskeleton of transformed cells. pp60^src was found to be localized on actin microfilaments present in adhesion plaques, at adherens junctions between cells and also in microfilament bundles. This localization is consistent with the hypothesis that some of the morphological effects of transformation result from the interaction in situ of pp60^src with microfilament-bound target proteins.     

Intercellular communication and the control of growth: XI. Alteration of junctional permeability by thesrc gene in a revertant cell with normal cytoskeleton

Summary To learn whether the reduction of cell-to-cell communication in transformation is a possible primary effect of pp60^src phosphorylation or secondary to a cytoskeletal alteration, we examined the junctional permeability in transformed cells with normal cytoskeleton. The permeability to fluorescentlabelled mono- and diglutamate was compared in clones of Faras' vole cells—clones transformed by Rous sarcoma virus and reverted from that transformation. One revertant clone (partial revertant), had the high levels of pp60^src kinase activity and tumorigenicity of the fully transformed parent clone, but had lost the cytoskeletal alterations of that clone. Another revertant clone (full revertant) had lost the tumorigenicity and most of the pp60^src kinase activity, in addition (J.F. Nawrocki et al., 1984,Mol. Cell Biol. 4:212). The junctional permeability of thepartial revertant with normal cytoskeleton was similar to that of the fully transformed parent clone with abnormal cytoskeleton. The permeabilities of both were lower than those of thefull revertant and the normal uninfected cell, demonstrating that the junctional change by thesrc gene is independent of the cytoskeletal one.     

Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus

All cells transformed by Rous sarcoma virus contain levels of phosphotyrosine in protein which are 6–10 fold greater than the very low levels present in uninfected cells. The increase is due largely to modification of cellular polypeptides. The abundance of phosphorylated tyrosines in protein in cells infected with tsLA29, a mutant of Rous sarcoma virus which is temperature-sensitive for cellular transformation, increases to 60% of maximum within 60 min of a shift to the permissive temperature and drops to a level close to that in uninfected cells within 60 min of a shift to the restrictive temperature. In light of the fact that pp60^src phosphorylates tyrosine in vitro, these results suggest strongly that the modification of one or more cellular polypeptides by way of pp60^src is critical for cellular transformation by Rous sarcoma virus. There is, however, no increase in the abundance of phosphotyrosine in protein in mouse cells transformed by Kirsten sarcoma virus, Moloney sarcoma virus, or SV40 virus, in chick embryo cells infected with avian myelocytomatosis virus MC29, and in rat and hamster cells transformed by polyoma virus. Thus increased phosphorylation of tyrosine is neither a universal mechanism of transformation nor an inevitable secondary cellular response to transformation.     

Fetotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for rhesus macaques (Macaca mulatta)

The fetotoxic and teratogenic potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for rhesus macaques (Macaca mulatta) was tested through oral administration to monkeys early in pregnancy. A single or divided dose, 1 μg of TCDD/kg of body weight, was followed by abortion in 13 of 16 pregnant monkeys treated between days 20 and 40 of gestation. One of four aborted at 0.2 μg/kg, and two of two at 5 μg/kg. None of the mothers given 0.2 μg/kg showed signs of toxicity. Eight of the monkeys aborting at 1 μg/kg showed clinical toxicity 44 to 111 days after aborting, and three died. Both given 5 μg/kg became toxic soon after abortion and died. No malformations except for two minor palatal abnormalities of questionable significance were found in the six fetuses that were not aborted at doses of 0.2 and 1.0 μg/kg. These results indicate (1) that TCDD is fetotoxic at doses that frequently have delayed toxicity to the mother, but that conclusions about teratogenicity cannot be drawn, and (2) that pregnant rhesus females are more sensitive to the toxic effects of TCDD than any species tested but the guinea pig.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an increase in protein kinases growth hepatic associated with epidermal factor receptor in the plasma membrane

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), administered to male rats at a single intraperitoneal (IP) injection dose of 25 μg/kg causes down-regulation of epidermal growth factor (EGF) receptor in the plasma membrane of rat liver which starts after two days and continues throughout the experimental period (20 days). Using monoclonal antibody to EGF receptor, it was determined that TCDD-caused EFG receptor down-regulation in the rat liver was accompanied by increased protein kinase activity. Such an increase in the protein kinase activity involves, at least in part, an activation of protein tyrosine kinase. Examination of serum samples from control and treated rats revealed no detectable difference in the level of EGF itself or EGF receptor-reacting substances (eg, hormones and other growth factors). In vivo TCDD caused early eye opening and tooth eruption and poor body weight gain and hair growth in mouse neonates similar to those observed with exogenously administered EGE The results indicate that such EGF receptor–mediated effect of TCDD has some toxocilogical significance in vivo. Although TCDD causes significant reduction in [¹²⁵I]-EGF binding in the hepatic plasma membrane in susceptible strains of mice, it has only modest effects in tolerant strains. The results are consistent with the idea that the action of TCDD on the EGF receptor is mediated through the cytosoliclnuclear TCDD receptor, which is known to be regulated by the Ah locus.     

Induction of down-regulation of the kinase activities of Mek,p42Erk,p90RSK,and p63SAMK in chicken embryo fibroblast at the late stage of src-induced cellular transformation

Two distinct stages in regulation of protein kinases are detectable upon cellular transformation of CEF induced by pp60^v-src. Upon cellular transformation induced by ts v-src mutants, the kinase activities of Mek and p42^Erk are rapidly induced at the early stage and significantly decreased at the late stage of cellular transformation. In contrast, a novel p63^SAMK is partially activated at the early stage and is fully activated at the late stage of cellular transformation. However, p90^RSK is activated through the entire course of cellular transformation. In this study, I detect a transient down-regulation of p90^RSK activity that is inducible in cultures at the late stage of the src-induced cellular transformation by an increase of extracellular pH value from 7 to 8 and unidentified components in DMEM, but not in cultures which are at the early stage. Concomitant with down-regulation of p90^RSK activity, the kinase activities of Mek, p42^Erk, and p63^SAMK are also down-regulated. Blockage of down-regulation of p90^RSK activity by pretreatment of cells with different phosphatase inhibitors correlates with blockage of the down-regulation of either p42^Erk or p63^SAMK activity. Multiple pathways appear to involve in regulation of p90^RSK activity. The discrepancy in regulation of protein kinase activity between the early and late stages of cellular transformation induced by pp60^src may indicate a change in signaling cascades during the progress of cellular transformation. The induction of the down-regulation event in this study may provide a new approach to investigate the regulation not only of protein kinases but also phosphatases in transformed cells. © 1996 Wiley Liss, Inc.     

p59fyn and pp60 c-src modulate axonal guidance in the developing mouse olfactory pathway

The Src-family tyrosine kinases p59^fyn and pp60^c-src are localized on axons of the mouse olfactory nerve during the initial stages of axonal growth, but their functional roles remain to be defined. To study the role of these kinases, we analyzed the trajectory of the olfactory nerve in E11.5 homozygous null mutant mice lacking single src or fyn genes and double mutants lacking both genes. Primary olfactory axons of single and double mutants exited the olfactory epithelium and projected toward the telencephalon, but displayed differences in fasciculation. The fyn-minus olfactory nerve had significantly more fascicles than the src-minus nerve. Most strikingly, the primary olfactory nerve of src/fyn double mutants showed the greatest degree of defasciculation. These defects, identified by NCAM labeling, were not due to apparent changes in the size of the olfactory epithelium. With the exception of the src-minus mice, which had fewer fascicles than the wild type, no obvious differences were observed in coalescence of vomeronasal axons from mutant mice. The mesenchyme of the double and single mutants exhibited only subtle changes in laminin and fibronectin staining, indicating that the adhesive environment of the mesenchyme may contribute in part to defects in fasciculation. The results suggest that signaling pathways mediated by p59^fyn and pp60^c-src contribute to the appropriate fasciculation of axons in the nascent olfactory system, and comprise partially compensatory mechanisms for axonal adhesion and guidance. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 53–63, 1998     

Mutants of polyomavirus middle-T antigen

Polyomavirus middle-T antigen induces the transformation of established cell lines in culture and is known to interact with and/or modulate the activity of several enzymes (pp60^c.src, protein kinase C and phosphatidylinositol kinase) in vitro. This review is a compilation of the reported mutants of middle-T antigen and their biochemical and biological properties as they relate to the transformation event. The mutants of polyomavirus middle-T antigen have been previously classified phenotypically. Given the now large number of mutants, the classification presented here is based upon the position within the molecule. A model of middle-T is presented in which the protein is considered as consisting of three domains: a hydrophobic domain (the putative membrane-binding domain), the amino-terminal half of the molecule (the putative pp60^c.src-binding domain) and the intervening amino acids (the putative modulatory domain). A current model for the induction of transformation by polyomavirus middle-T is presented.     

2,3,7,8-tetrachlorodibenzo-p-dioxin mechanism of action to reduce lipoprotein lipase activity in the 3T3-L1 preadipocyte cell line

Lipoprotein lipase (LPL) is important in the process of triglyceride storage in adipose tissue. Depression of LPL activity in adipose tissue is associated with 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD)-induced wasting syndrome and may have a role in the associated serum hyperlipidemia produced by TCDD. The 3T3-L1 cell line was used as an in vitro model, independent of hormonal, nutritional, or other interfering factors associated with in vivo studies, in order to systematically examine the mechanism of action of TCDD. TCDD produced a statistically significant (P < 0.05) time- and dose-dependent decrease in LPL activity. Results of experiments with Ah-receptor blockers and structure activity studies with different polychlorinated biphenyl (PCB) and dioxin congeners were consistent with reduction of LPL activity being mediated by the Ah receptor. Culturing of 3T3-L1 cells without glucose or with cytochalasin B, a blocker of facilitative glucose transporters (GLUT), was effective in reducing LPL activity (P < 0.05). TCDD did not further reduce LPL activity in cytochalasin B pretreated 3T3-L1 cells or in 3T3-L1 cells cultured in glucose-free media. Dexamethasone pretreatment, which is known to increase GLUT expression in 3T3-L1 cells, prevented the reduction of LPL activity by TCDD. Protein tyrosine kinase activities, assayed using γ-³²P-ATP and RR-SRC, a src specific peptide substrate, were significantly increased (P < 0.05) over control levels by both TCDD and glucose deprivation. Furthermore, results of experiments treating 3T3-L1 cells with either insulin, EGF, 8-Br-cAMP, TPA, or genistein, alone or in combination with TCDD, were generally consistent with the hypothesis that lowered intracellular glucose and altered cellular kinase activities may be involved in reduction of LPL activities by TCDD. Further work is needed to confirm and better understand the role protein phosphorylation plays in TCDD-mediated alteration of glucose disposition and LPL activity. In summary, TCDD reduced LPL activity in 3T3-L1 cells as seen in vivo. Manipulation of glucose transport through a number of experimental approaches produced changes in 3T3-L1 LPL activity consistent with results of previous investigators showing glucose to be a positive regulator of LPL activity and consistent with our hypothesis that TCDD-mediated reduction of glucose transport is an important factor in the down regulation of LPL activity by TCDD. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 29–39, 1998     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters pancreatic membrane tyrosine phosphorylation following acute treatment

To understand the basic mechanisms of TCDD's action to cause hypoinsulinemia in several experimental animals, we have studied TCDD-induced changes in various protein kinase activities in membrane preparations of guinea pig pancreas. For this purpose, young male guinea pigs were treated through a single intraperitoneal in jection with 1 or 3 μg/kg of TCDD in vivo, and, after given time periods, pancreas samples were obtained and membranes were isolated through homogenization and centrifugation procedures. Several sets of incubation conditions were selected for protein kinase activity assay, each favoring a specific type of protein kinase. It was found that overall protein phosphorylation activities were higher in the preparation from TCDD-treated an imals as compared to those found in pair-fed controls and that this trend was more pronounced when the assay medium contained Mn²⁺ in place of Mg²⁺ and EGTA. These are the conditions that are known to favor protein tyrosine kinases. Other types of protein kinases from the treated animals did not show any significant differences from the pair-fed control animals, though that of protein kinase C in the treated preparation showed a modest increase. To establish that the type of protein kin ases stimulated by TCDD are protein tyrosine kin ases, we have carried out phosphoamino acid analyses, KOH digestion, and western blot analyses using an antibody to phosphotyrosine. All the results were consistent in supporting the idea that TCDD causes a rise in protein-tyrosine kinases in pancreas at early stages of poisoning.