Search

Background: One of the outcomes positively associated with dioxin exposure in humans is type 2 diabetes., Objectives: This study was conducted in order to find the molecular biological evidence for the diabetogenic action of dioxin in adipose samples from Vietnam veterans., Methods: We obtained 313 adipose tissue samples both from Vietnam veterans who were exposed to dioxin (Operation Ranch Hand) and from comparison veterans who served in Southeast Asia with no record of dioxin exposure. We conducted quantitative reverse-transcribed polymerase chain reaction studies on selected marker mRNAs from these samples., Results: We found the most sensitive and reliable molecular indicator of dioxin-induced diabetes to be the ratio of mRNA of glucose transporter 4 (GLUT4) and nuclear transcription factor kappa B (NFkappaB), a marker of inflammation. This ratio showed significant correlations to serum dioxin residues and to fasting glucose among those in the Ranch Hand group and, surprisingly, even in the comparison group, who have low levels of dioxin comparable to the general public. Such a correlation in the comparison group was particularly significant among those with known risk factors such as obesity and family history of diabetes., Conclusions: These results show that the GLUT4:NFkappaB ratio is a reliable marker for the diabetogenic action of dioxin, particularly at very low exposure levels that are not much higher than those found in the general public, implying a need to address current exposure levels.

The most consistent toxic effects of dioxin-type chemicals are hyperlipidemia, body weight loss (particularly body fat loss), anorexia, changes in carbohydrate metabolism, and lipid peroxidation. The biochemical systems particularly affected are lipoprotein lipases, low-density-lipoprotein receptors, glucose transporter proteins (GLUTs), vitamin C uptake, and insulin secretion. Some of these biochemical changes occur at very low doses, and some effects can last for long time periods. To provide a mechanistic explanation for such actions of dioxins, available experimental evidence has been reviewed. The most recent discovery indicates that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) directly acts with isolated cytosolic aryl-hydrocarbon (Ah) receptor under cell-free conditions even without the presence of the nucleus and is capable of activating key protein kinases that are involved in the growth factor signal-transduction pathway. The resulting activation of primary-response transcription factors in the nucleus appears to play a key role in coordinating vital cell program shifts, including lipid metabolism.

Within 1 hr of intraperitoneal administration of 1 microgram 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg, lipoprotein lipase (LPL) activity was reduced 38% from initial levels in the adipose tissue of the guinea pig. Maximal depression was observed after 2 days and persisted throughout the 10-day observation period. Oral administration of glucose restored LPL activity in TCDD-treated animals after 1 day but only partially after 2 and 5 days, and had no effect after 10 days of exposure. Although initial (2-day) serum insulin levels were depressed, the inability of glucose to restore LPL activity after prolonged exposure was not due to malabsorption of glucose nor to changes in serum thyroxine or insulin concentration. TCDD also inhibited the lipolytic pathway in the adipocyte, but had no effect on hormone sensitive lipase (HSL). Since HSL and LPL are reciprocally regulated, it was concluded that TCDD acts on the adipocyte to uncouple HSL-LPL reciprocity as well as to reduce LPL production.

Administration of 1 micrograms/kg (single intraperitoneal injection and studied after 10 days) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to young male guinea pigs was found to cause a significant reduction in binding of low-density lipoprotein (LDL) to its receptor on the hepatic plasma membrane. This reduction in LDL binding is not caused by the decrease of food intake by treated animals since pair-fed control animals had significantly higher LDL binding than treated animals. It was also found that primary hepatocytes from treated animals had a reduced ability to internalize LDL than controls. Such a change in the plasma membrane function may explain the resulting hyperlipidemia particularly hypercholesterolemia which occurs in this species as a result of TCDD administration.

The effect of in vivo-administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF) receptor activity of the rat hepatic plasma membrane was studied. TCDD causes a significant reduction in EGF binding at an early stage of toxicity (day 2) and at very low doses (1 microgram/kg, single i.p., rat). This reduction appears to be due to a decline in the number of receptors. There is a good correlation between levels of decline in EGF binding and loss of body weight among TCDD-treated rats. The reduction in EGF binding occurs at a relatively low dose in the guinea pig (a very sensitive species) and at high doses in the hamster (a tolerant species). Among three mice strains, TCDD (115 micrograms/kg, single i.p.) caused 98% reduction in EGF binding in the sensitive strains (C57BL/6J and CBA/J) but only a 50% reduction in the tolerant strain (AKR/J). To relate the above biochemical changes to in vivo effects, TCDD was postnatally administered (through mother's milk) to mouse neonates. The most prominent toxic manifestations were early eye opening and incisor eruption, loss in body weight gain, and retardation of hair growth. All of these symptoms have been ascribed to EGF effects. TCDD was also found to stimulate phosphorylation of the EGF receptor in the rat hepatic plasma membrane. This phosphorylation effect was observed at day 1 and persisted until the end of the test (day 10). It has long been recognized that agents causing reduction in number of EGF receptors (e.g., phorbol esters) elicit in vivo cellular responses that are similar to those caused by exposure to excess doses of growth factors. Accordingly, a hypothesis has been proposed to ascribe some of the EGF-like effects of TCDD, such as fatty infiltration of the liver and hyperplastic proliferation of gastric epithelia and epidermal cells to its action on the EGF receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been found to cause increases in cellular levels of pp60src, a protein tyrosine kinase in hepatocytes from the rat and guinea pig, in the thymus of the mouse in vivo and in NIH-3T3 mouse fibroblast cell lines in vitro. Such cellular changes take place in vivo at early stages of TCDD poisoning (as early as one day after treatment in the case of mouse thymus) and at very low doses (single intraperitoneal injections of 1 microgram/kg for guinea pigs, 25 micrograms/kg for rats, and 30 micrograms/kg for mice). In addition such an effect of TCDD was observed only in a TCDD-responsive mouse strain but not in a nonresponsive strain. This effect of TCDD is a long-lasting one (eg, even 25 days after single dosing, the levels of pp60src in the hepatic membrane remained high). In vitro this effect was observed in a wild-type 3T3 cell line but was more pronounced in one of the transfected lines with a v-src gene, a virus-derived oncogene known to code for pp60src protein.

Serum insulin and glucose were measured in young male rabbits after a single intraperitoneal dose of 1 or 50 micrograms/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Serum insulin levels in the high dosed rabbits were significantly decreased between 15 min and 8 h post treatment, equivalent after 24 h, significantly elevated 48 h post treatment, and they were not different at 10 days post-treatment when compared to weight matched and pair-fed controls. At the low dose, rabbits showed no differences in serum insulin from controls. In the high dose group, serum glucose levels were generally not different between treated and control animals, though there was a transient hyperglycemia 1 h after treatment, and both treated groups became hypoglycemic after ten days. The results indicate that TCDD altered serum insulin levels which were not coupled to changes in serum glucose.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) caused a dose-dependent decrease of adipose tissue lipoprotein lipase (LPL) activity and caused a concomitant increase in serum triglyceride concentration in the rabbit 10 d after single ip administration of either 1 or 50 micrograms/kg. Hepatic low-density lipoprotein (LDL) binding was markedly depressed and serum cholesterol concentrations were modestly increased relative to pair-fed control animals. Serum glucose concentrations were significantly lower in the rabbit administered TCDD compared to ad libitum or pair-fed control animals, although little change was observed in serum insulin concentration. Electron microscopic examination of aortic arches 20 d after a single ip administration of 50 micrograms TCDD/kg revealed ruffling, denudation, and sloughing off of the cell surface and the appearance of macrophage-like structures in the intima and media of the endothelial cells. These alterations resemble preatherosclerotic lesions typical in animals with hyperlipidemia. It is proposed that TCDD causes hyperlipidemia in the rabbit through suppression of LPL activity and LDL receptor binding.

TCDD was found to cause a marked inhibition of 125I-epidermal growth factor (EGF) binding to its receptor on the cell surface of XB mouse keratinizing epithelial cells (XB cells) cultured in vitro. The EC50 concentration was estimated to be on the order of 3 x 10(-11) M 24 hours after TCDD administration. As early as 12 hours after the addition of 10(-9) M of TCDD, XB cells showed signs of a decline in 125I-EGF binding levels. The level of such EGF receptor downregulation reached a maximum at 24 hours, continued until day 2, but completely recovered by day 3. This was accompanied by a rise in protein kinase activities, particularly those of the protein tyrosine kinases during the initial period of 6-24 hours. To test the hypothesis that the EGF receptors of the cells, by showing TCDD-induced symptoms of downregulation, actually are being activated and triggering EGF-like signals, we examined the effects of both TCDD and exogenously added EGF on cell morphology, colony formation degree of keratinization, the pattern of activation of protein kinases and de novo protein synthesis, and EGF receptor phosphorylation. Based on the similarity of cell responses to these between TCDD- and EGF-treated cells, we concluded that TCDD, directly or indirectly, causes activation of the EGF receptor. In contrast, 12-O-tetradencanoylphorbol-13-acetate (TPA), which is known to downregulate EGF receptors by blocking their protein tyrosine kinase, produced dissimilar end results. The balance of evidence support the notion that the action of TCDD in this cell line is tightly coupled to the activation of the EGF receptor and that one of the key consequences of such a biochemical change is that it signals these cells to commit to terminal differentiation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), administered to male rats at a single intraperitoneal (IP) injection dose of 25 micrograms/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 EGF. 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 cytosolic/nuclear TCDD receptor, which is known to be regulated by the Ah locus.

TCDD was found to cause a significant rise in protein tyrosine kinase levels at an early stage of poisoning in rat liver membrane preparations. The results of sephadex G-150 column chromatographic analysis on rat hepatocyte membranes indicate that there are at least three tyrosine kinases of which activities increase as a consequence of TCDD treatment in vivo. The TCDD-evoked rise in such protein tyrosine kinase activity precedes the down-regulation of the EGF (epidermal growth factor) receptor in the plasma membrane in vivo.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) administered to young male guinea pigs at a dose of 1 microgram/kg (single intraperitoneal injection) caused a large reduction in adipose tissue lipoprotein lipase (LPL) activity. This effect occurred rapidly; a 70% decrease was noticed after 24 hour and 80% of LPL activity was lost by 48 hours when the serum triglyceride levels increased to 175% of control levels. LPL is known to play an important role in controlling the amount of free fatty acids supplied to adipose tissues. Administration of a large dose of glucose to fasted guinea pigs, which have shown a similar weight loss, but less LPL loss than TCDD-treated animals, had the effect of elevating their adipose LPL levels back to a near normal level, whereas the same treatment caused no significant increase in the LPL levels of TCDD-treated animals. Evidence indicates that the TCDD-caused decline in LPL activity is irreversible. As a consequence, the affected guinea pigs are incapable of responding to changes in nutritional status.

Cardiac effects of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) were examined using atrial muscle isolated from young or mature guinea pigs fed ad libitum, from young animals 10 or 20 days after a single ip injection of 1 microgram TCDD/kg body wt, and from control guinea pigs pair-fed to the TCDD-treated animals. Basal contraction force of left atrial muscle obtained from TCDD-treated and pair-fed control guinea pigs 10 days after treatment was significantly increased compared to atrial muscle obtained from ad libitum control animals. Atrial muscle from TCDD-treated animals 20 days after treatment had a significantly lower basal force of contraction while that from pair-fed animals was significantly higher relative to atrial muscle from ad libitum control animals. These results suggest that the TCDD-induced change in basal atrial contraction force is in part due to undernutrition and that prolonged exposure to TCDD inhibits this response observed in pair-fed animals. Furthermore, prolonged TCDD treatment shifted the dose-response curve for the positive inotropic effect of isoproterenol to the right with no statistically significant change in the maximal inotropic effect obtained with high concentrations of isoproterenol. The TCDD-induced changes in force of contraction and the dose-response curve for the positive inotropic effect of isoproterenol were similar in young TCDD-treated and mature control guinea pigs. TCDD did not alter the basal contraction frequency or the dose-response curve for the positive chronotropic effect of isoproterenol. There was no effect on cardiac lipoprotein lipase activity at 1 microgram/kg, but in animals administered 4 micrograms TCDD/kg activity was reduced. It was concluded that TCDD adversely affects atrial muscle of guinea pig heart.

1. Adipose tissue lipoprotein lipase (LPL) activity of several different animal species was determined after i.p. administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 2. TCDD caused a significant reduction in LPL activity and an increase in serum triglyceride concentration in guinea pigs, rabbits, and hamsters but not rats. 3. TCDD increased adipose tissue LPL activity of mink and lowered their serum triglyceride concentration. 4. Results of this study indicate that profound differences occur in lipid metabolism between various species in response to TCDD and these changes do not appear to be related to generalized toxicity such as wasting.

The fate of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) was studied by using aquatic sediment and lake water under laboratory conditions. Most of the TCDD was found in the sediment from which it slowly disappeared. Evaporation may be a major mode of disappearance of TCDD in samples incubated 39 days or more, with metabolism playing only a minor role. Under the experimental conditions the half-life of TCDD was in the order of 600 days. The metabolic activities were enhanced under conditions which stimulated microbial growth in the presence of sediment, and the metabolites were found to be released from the sediment to the ambient water.

2,3,7,8,-Tetrachlorodibenzo-p-dioxin (TCDD) administered in vivo causes drastic reduction in the weight of the mouse thymus at low doses (e.g., 30 micrograms/kg single i.p. injection), the reduction becoming statistically significant after 2 days. To understand the cause for such thymic involution TCDD-evoked changes in various biochemical parameters in this tissue were examined. The most noticeable change was observed in the increased activity of specific protein-tyrosine kinases and protein kinase C and an increased level of p21ras-associated binding of [3H]GTP. Since no significant change was observed with cAMP-stimulated protein kinases and cAMP levels, the above changes appear to be a selective effect on these special classes of proteins. As a result of a time sequence study it has become apparent that the rise in protein-tyrosine kinase activities becomes significant within 24 hr, whereas the rise in protein kinase C does not become significant until 48 hr. Among protein-tyrosine kinases, pp60c-src and probably pp561skT were found to be significantly elevated by TCDD treatment. In view of similarities between TCDD and thyroid hormones in causing thymic involution, the levels of c-erb-A expression were assessed in the liver by using avian 32P-labeled v-erb-A probe and RNA transfer blot hybridization technique. The results clearly indicate that TCDD has the property to elevate levels of mRNA bearing homology to v-erb-A. Such changes in c-erb-A expression and protein-tyrosine kinase occurred only in TCDD-susceptible (responsive) strains but not in tolerant (nonresponsive) strains of mice at the dose tested. Based on such observations a hypothesis has been proposed that TCDD owes its potency to its ability to stimulate the expression of one of a family of DNAs bearing homology to v-erb-A and that one of the major consequences of such an action is stimulation of various tyrosine kinases.

To study the cause of TCDD-evoked changes in the functions of plasma membrane constituents TCDD's effects on protein kinase activities in the liver of rats and guinea pigs were investigated. TCDD was found to cause a sharp increase in both c-AMP independent and dependent protein kinase activities in plasma membrane preparations from rat liver within 48 hours from the time of administration. Such effects reached maxima around day 20, and were quite noticeable even 40 days after a single administration of TCDD. As a result of SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) analysis several substrate proteins for these increased protein kinases were observed. Among them are 170 K - 150 K bands, representing EGF receptor protein. TCDD was found to particularly stimulate protein kinase C which is known to influence many enzyme and receptor functions through protein phosphorylation. The possible significance of such an action of TCDD is discussed.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF)-binding characteristics was studied in a cultured embryonic fibroblast cell line, C3H 10T1/2. At very low concentrations, TCDD was found to cause a persistent decline in EFG binding, the median effective concentration (EC-50) being 10(-12) M. This particular effect was most conspicuous when TCDD was added at the time of medium change with fresh Dulbecco's modified Eagle's medium. Cells at an early stage of confluency were more responsive to TCDD than those at a later stage. Although most reported TCDD-evoked biological changes are recognized to occur slowly during the course of a few days to weeks, the response of C3H 10T1/2 cells to TCDD was swift, showing a sign of decline of EGF binding as early as three hours after TCDD addition. C3H 10T1/2 cells appear to be an excellent in vitro model to study TCDD's biochemical action mechanisms.