Retrospective analysis of carcinogenicity assessments within FDA-notified GRAS determinations.

Frameworks have been developed to standardize the assessment of carcinogenic potential in the pharmaceutical and agrochemical industries, building upon decades of research. Carcinogenicity is also evaluated during the safety evaluation of food substances, using a comprehensive approach unique to each substance. To better understand these approaches, a retrospective assessment was conducted on the publicly available database of substances notified to the United States Food and Drug Administration (US FDA) as being Generally Recognized As Safe (GRAS). The data contained within these GRAS notifications (GRNs) were reviewed for the methods used to evaluate carcinogenic potential (genotoxicity studies, 2-year bioassays, other pre-clinical animal studies) to identify patterns that could provide an understanding of how this assessment has been conducted for different categories of food substances. While different approaches to the safety evaluation were required to adapt to the unique food substances, the data in all notifications supported the conclusion of safety. The evaluation of food substances for carcinogenic potential must consider all available data, including identifying the need for when more data must be generated to support an evaluation. Due to the complexity of substances used in food, ranging from defined chemical entities to minimally processed agricultural commodities to live microorganisms, the approach to conducting the safety evaluation of food substances must be able to adapt to the most relevant scientifically supported approach. This paper illustrates the data commonly used to support the safety of different types of food substances and proposes an approach familiar to other product sectors.

An evolution of risk assessment for potential carcinogens in food: Scientific session proceedings.

Modern perspectives on the risk assessment of carcinogenic potential of chemicals have taken shape within the last two decades. This has been due to both developments in the understanding of the biology and etiology of cancer and by advances in in silico and in vitro assays. Moving away from a conventional binary carcinogen/non-carcinogen model, modern frameworks offer more nuanced classification structures based on the understanding of mechanisms involved or potentially involved in rodent carcinogenicity. Given these developments, a scientific session at the 2020 Winter Meeting of the Toxicology Forum was organized to explore the impact these innovative approaches will have on food safety assessments and what considerations should be addressed in developing a new carcinogenic risk assessment approach for substances in foods. The session reviewed challenges faced by food toxicologists and risk assessors, current standard approaches for evaluating carcinogenic risk of food substances, limitations of these standard approaches, and potential methods to implement next generation assays and modern carcinogenic frameworks into food safety assessments. Current perspectives of US regulatory, industry, and academic stakeholders were represented during speaker presentations and a moderated Panel Discussion. This Workshop Report provides an overview of key themes and information presented during the session. Summary statements were prepared by the authors and reviewed by the presenters but do not necessarily represent the position or policy of the FDA, the EPA, or other affiliations.

A 3-week pre-clinical study of 2'-fucosyllactose in farm piglets.

One of the most abundant oligosaccharides found in human milk is 2'-fucosyllactose, a trisaccharide composed of fucose and lactose, and multiple studies have demonstrated a health benefit to this compound. Recent advances have allowed for the large-scale production of oligosaccharides via fermentation, including 2'-fucosyllactose. A neonatal piglet model was used to evaluate the tolerability of 2'-fucosyllactose, produced through this process, in order to demonstrate the suitability of this compound for human infants under 12 weeks of age. Crossbred farm piglets, at lactation day 2, were assigned to one of four treatment groups receiving a liquid diet containing 0, 200, 500 or 2000 mg/L of 2'-fucosyllactose. The calculated consumption of 2'-fucosyllactose corresponded to dose levels of 29.37, 72.22 and 291.74 mg/kg/day, respectively, in males and 29.30, 74.31, and 298.99 mg/kg/day, respectively in females. Piglets were administered diet for 3 weeks; and there were no test article-related effects on growth and development (clinical observations, body weight and food consumption), clinical pathology parameters (hematology, clinical chemistry, coagulation and urinalysis), or any histopathologic changes. Therefore, dietary exposure to 2'-fucosyllactose at concentrations up to 2000 mg/L was well tolerated by neonatal farm piglets and did not result in adverse health effects or impact piglet growth.

Spanish black radish (Raphanus sativus L. Var. niger) diet enhances clearance of DMBA and diminishes toxic effects on bone marrow progenitor cells.

Vegetables of the Cruciferae family contain high levels of glucosinolates, metabolites of which are believed to enhance detoxification. Spanish black radishes (SBR) contain 4× more glucosinolates than other crucifers. This study examined whether feeding mice a diet containing 20% SBR for 2 wk could enhance metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) and inhibit DMBA-mediated bone marrow toxicity. Expression of Phase I and II detoxification enzymes was significantly greater for mice fed SBR than control diet. Six hours after DMBA administration, the blood levels of DMBA in mice fed the SBR diet were significantly lower than mice fed a control diet. DMBA reduced bone marrow cells in mice fed control diet to a significantly greater extent than mice fed the SBR diet. Colony forming assays demonstrated that mice on the SBR diet had 1) less reduction in lymphoid CFU-preB progenitor cells, 2) greater recovery of CFU-preB progenitor cells at 168 h, and 3) less reduction of CFU-GM progenitor cells at 6 h. Therefore, mice fed a 20% SBR diet for 2 wk had greater expression of detoxification enzymes, faster metabolism of DMBA, and a reduction in DMBA-induced bone marrow toxicity. Overall, these results support the hypothesis that glucosinolates in SBR are protective against acute toxicity.

Phytochemical composition and biological activity of 8 varieties of radish (Raphanus sativus L.) sprouts and mature taproots.

Radishes (Raphanus sativus L.) are members of the cruciferous vegetable family that contain many classes of biologically active phytochemicals. This study determined the phytochemical composition of the sprouts and mature taproots of 8 radish varieties. Radish sprouts contained significantly greater concentrations of glucosinolates (3.8-fold) and isothiocyanates (8.2-fold) than the mature radish taproot and also contained significantly greater concentrations of phenolics (on average 6.9-fold). The anthocyanin concentrations of the mature radish taproot were significantly greater than in the sprouts of red, pink, and purple varieties. The primary anthocyanidins present in the red and pink radish varieties were pelargonidin and delphinidin, while the primary anthocyanidin in the purple radish variety was cyanidin. Radish sprouts were between 9- and 59-fold more potent than the corresponding mature taproot at activating the antioxidant response element (ARE) in a stably transfected hepatoma cell line. The ARE activity of the radish sprouts and mature taproots was significantly correlated with the total isothiocyanate concentration of the radishes. Practical Application: Understanding the influence variety and developmental stage has on the biological activity of cruciferous vegetables provides important information for further studies examining the in vivo effects of radish treatment and foundation for providing recommendations to reduce the risk of chronic disease through dietary intervention.

Heat treatment of Brussels sprouts retains their ability to induce detoxification enzyme expression in vitro and in vivo.

UNLABELLED: The bioactive metabolites of glucosinolates, such as isothiocyanates, contained in cruciferous vegetables have been shown to reduce the risk of cancers through the induction of detoxification enzymes. However, cruciferous vegetables are commonly processed before consumption, significantly altering the phytochemical composition of these vegetables. Compared to freeze-dried Brussels sprouts, oven-dried Brussels sprouts contain low concentrations of glucosinolates (22.14 and 0.85 µmol/g, respectively) and isothiocyanates (3.68 and 0.15 µmol/g, respectively). The effect of oven-dried Brussels sprouts on the expression of detoxification enzymes was evaluated in vitro and in vivo. Treatment of immortalized human hepatoma cells with the aqueous extract from oven-dried Brussels sprouts significantly increased quinone activity (0.5 and 1.5 mg/mL) and the activity of the antioxidant response element (EC50=2.39 mg/mL) and xenobiotic response element (EC50 2.92 mg/mL). C3H/HeJ mice fed a diet containing 20% oven-dried Brussels sprout diets for 2 wk demonstrated significantly higher expression than animals fed a nutrient-matched control diet of CYP1A1, CYP1A2, and epoxide hydrolase in the liver and CYP1A1, CYP1A2, CYP1B1, epoxide hydrolase, UGT1A1, thioredoxin reductase, and heme oxygenase in the lungs. The low concentrations of glucosinolates and isothiocyanates in oven-dried Brussels sprouts suggest that other compounds, such as the Maillard reaction products that are produced during heating, are responsible for the induction of detoxification enzymes in vitro and in vivo. PRACTICAL APPLICATION: The manner in which cruciferous vegetables are processed prior to consumption has significant effects on what compounds people are exposed to. The presence of glucosinolates or isothiocyanates can be a good indicator of the ability of cruciferous vegetables to induce detoxification enzymes. However, the data presented here demonstrate that while heat processing of Brussels sprouts greatly reduced the concentrations of glucosinolates and isothiocyanates, their ability to induce detoxification enzymes in vitro and in vivo was retained.

Raphasatin is a more potent inducer of the detoxification enzymes than its degradation products.

UNLABELLED: The biological activity of cruciferous vegetables is hypothesized to be due to the metabolites of a class of phytochemicals called glucosinolates. The chemical properties of these metabolites, including isothiocyanates, determine the biological activity of these compounds and thus their effects on human health. The 2 primary radish (Raphanus sativus L.) glucosinolates, glucoraphasatin, and glucoraphenin, were isolated using solid phase extraction followed by preparative HPLC purification. In an aqueous environment, 77.6% of the maximum amount of sulforaphene produced by the metabolism of glucoraphenin was present after 24 h. Under the same conditions raphasatin, the isothiocyanate metabolite of glucoraphasatin and the oxidized counterpart of sulforaphene, was highly unstable with a half-life of less than 30 min and no raphasatin was detectable after 24 h. In HepG2 cells, raphasatin-induced quinone reductase activity and the RNA expression of several phase 1 and 2 detoxification enzymes by a significantly greater amount than the degradation products of raphasatin. Raphasatin, but not its degradation products, activated the antioxidant response element (ARE) in a stably-transfected reporter cell line. Mice fed a diet consisting of 20% freeze dried radishes for 2 wk had significantly higher liver expression of cytochrome P450 (CYP) 1A1, 1A2, quinone reductase, microsomal epoxide hydrolase, and glutathione S-transferase α2 than mice fed a nutritionally-matched control diet. PRACTICAL APPLICATION: Glucoraphasatin, the primary glucosinolate in radishes, is metabolized into an isothiocyanate (raphasatin) that has biological activity but is also unstable in an aqueous environment. Despite the instability of raphasatin, dietary exposure to radishes produced significant induction of detoxification enzymes. Understanding the chemical properties of raphasatin, both in terms of biological activity and instability, could help develop processing methods to retain the most activity from radishes, glucoraphasatin, and raphasatin.

Induction of detoxification enzymes by feeding unblanched Brussels sprouts containing active myrosinase to mice for 2 wk.

In cruciferous vegetables, myrosinase metabolizes the relatively inactive glucosinolates into isothiocyanates and other products that have the ability to increase detoxification enzyme expression. Thus, maintaining myrosinase activity during food preparation may be critical to receiving the maximum benefit of consumption of Brussels sprouts or other cruciferous vegetables. To test the importance of maintaining myrosinase activity for maximizing bioactivity, experimental diets containing 20% unblanched (active myrosinase) or 20% blanched (inactivated myrosinase) freeze-dried Brussels sprouts and a nutrient-matched control diet were evaluated in vitro and in vivo for their ability to induce detoxification enzymes. Treatment of immortalized HepG2 human hepatoma cells with the unblanched Brussels sprout diet caused a greater increase quinone activity compared to the blanched Brussels sprout diet. C3H/HeJ mice fed the unblanched Brussels sprout diets for 2 wk had significantly higher plasma sulforaphane concentrations. Liver expression of CYP1A1 and epoxide hydrolase, measured using real-time PCR, was correlated with the plasma concentration of sulforaphane. In the lung, expression of epoxide hydrolase, thioredoxin reductase, UDP glucuronosyltransferase, quinone reductase, heme oxygenase, CYP1A1, CYP1A2, and CYP1B1 were also correlated with the plasma concentration of sulforaphane. Together these data demonstrate that, as predicted by the in vitro experiment, in vivo exposure to Brussels sprouts with active myrosinase resulted in greater induction of both phase I and phase II detoxification enzymes in the liver and the lungs that correlated with plasma sulforaphane concentrations.

Aqueous extracts from dietary supplements influence the production of inflammatory cytokines in immortalized and primary T lymphocytes.

BACKGROUND: Congaplex and Immuplex are dietary supplements that have been traditionally used to support immune system function. The purpose of these experiments was to determine whether Congaplex and Immuplex affect immune function using primary and immortalized T lymphocytes. METHODS: Immortalized CEM and Jurkat T lymphocytes and primary peripheral mononuclear blood cells (PBMCs) were treated with the aqueous extracts from Congaplex and Immuplex to determine the effects of these products on cytokine production in activated T lymphocytes. RESULTS: Congaplex enhanced phytohemagglutinin/phorbol 12-myristate 13-acetate (PHA/PMA) stimulation of both CEM and Jurkat cells as measured by the production of cytokines, while Immuplex suppressed PHA/PMA-induced production of cytokines, with the exception of interleukin (IL)-8 which was enhanced by Immuplex. In vitro treatment of PBMCs from 10 healthy donors with Congaplex or Immuplex decreased PHA-stimulated production of interferon (IFN)-gamma but increased the production of IL-13. CONCLUSIONS: While the effects of Congaplex and Immuplex differed in these two models, these data demonstrate that the aqueous extracts from these two dietary supplements can affect the inflammatory response of T lymphocytes.

Aqueous extract from Spanish black radish (Raphanus sativus L. Var. niger) induces detoxification enzymes in the HepG2 human hepatoma cell line.

Spanish black radish (Raphanus sativus L. var. niger) is a member of the Cruciferae family that also contains broccoli and Brussels sprouts, well-known to contain health-promoting constituents. Spanish black radishes (SBR) contain high concentrations of a glucosinolate unique to the radish family, glucoraphasatin, which represents >65% of the total glucosinolates present in SBR. The metabolites of glucosinolates, such as isothiocyanates, are implicated in health promotion, although it is unclear whether glucosinolates themselves elicit a similar response. The crude aqueous extract from 0.3 to 3 mg of dry SBR material increased the activity of the phase II detoxification enzyme quinone reductase in the human hepatoma HepG2 cell line with a maximal effect at a concentration of 1 mg/mL. Treatment of HepG2 cells with the crude aqueous extract of 1 mg of SBR per mL also significantly induced the expression of mRNA corresponding to the phase I detoxification enzymes: cytochrome P450 (CYP) 1A1, CYP1A2, and CYP1B1 as well as the phase II detoxification enzymes: quinone reductase, heme oxygenase 1, and thioredoxin reductase 1. Previous studies have shown that the myrosinase metabolites of different glucosinolates vary in their ability to induce detoxification enzymes. Here, we show that while glucoraphasatin addition was ineffective, the isothiocyanate metabolite of glucoraphasatin, 4-methylthio-3-butenyl isothiocyanate (MIBITC), significantly induced phase II detoxification enzymes at a concentration of 10 microM. These data demonstrate that the crude aqueous extract of SBR and the isothiocyanate metabolite of glucoraphasatin, MIBITC, are potent inducers of detoxification enzymes in the HepG2 cell line.

Microarray analysis of early adipogenesis in C3H10T1/2 cells: cooperative inhibitory effects of growth factors and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

C3H10T1/2 mouse embryo fibroblasts differentiate into adipocytes when stimulated by a standard hormonal mixture (IDMB). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), via the aryl hydrocarbon receptor (AhR), inhibits induction of the key adipogenic gene peroxisome proliferator-activated receptor gamma (PPARgamma) and subsequent adipogenesis. This TCDD-mediated inhibition requires activation of the extracellular signal-regulated kinase (ERK) pathway, which can be accomplished by serum, epidermal growth factor (EGF), or fibroblast growth factor (FGF). In the absence of serum or growth factors, IDMB induced adipogenesis without mitosis. Microarray analysis identified 200 genes that exhibited expression changes of at least twofold after 24 h of IDMB treatment. This time precedes most PPARgamma stimulation but follows the period of TCDD/ERK cooperation and periods of increased cell contraction and DNA synthesis. Functionally related gene clusters include genes associated with cell structure, triglyceride and cholesterol metabolism, oxidative regulation, and secreted proteins. In the absence of growth factors TCDD inhibited 30% of these IDMB responses without inhibiting the process of differentiation. A combination of EGF and TCDD that blocks differentiation cooperatively blocked a further 44 IDMB-responsive genes, most of which have functional links to differentiation, including PPARgamma. Cell cycle regulators that are stimulated by EGF were substantially inhibited by IDMB but these responses were unaffected by TCDD. By contrast, TCDD and EGF cooperatively reversed IDMB-induced changes in cell adhesion complexes immediately prior to increases in PPARgamma1 expression. Changes in adhesion-linked signaling may play a key role in TCDD affects on differentiation.

Differentiation of pluripotent C3H10T1/2 cells rapidly elevates CYP1B1 through a novel process that overcomes a loss of Ah Receptor.

Stimulation of C3H10T1/2 cells by an adipogenic hormonal mixture (IDM) consisting of insulin (I), dexamethasone (D), and methylisobutylxanthine (M) substantially induces cytochrome P450 (CYP) 1B1 expression. This stimulation represents up to 40% of the level produced by maximum activation of the arylhydrocarbon receptor (AhR) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dexamethasone and methylisobutylxanthine in combination produced near maximum elevation of CYP1B1 along with a subsequent decline in AhR that paralleled the rise in peroxisome proliferator-activated receptorgamma1 (PPARgamma1). Inhibitors of AhR activity, which block TCDD induction, did not affect this increase of CYP1B1 expression, which was, therefore, independent of AhR activity. These responses were unaffected by inhibition of DNA synthesis, which was required for PPARgamma1 induction and terminal differentiation. Induction of CYP1B1 mRNA was paralleled by increased CYP1B1 promoter-luciferase reporter activity. The initial 0.8kb of promoter region, which was sufficient for 24h near maximum stimulation, did not contain either the key AhR-responsive elements that mediate the TCDD response or CREB and SF1 elements that mediate cAMP stimulation of rat CYP1B1 in steroidogenic cells. This reporter response to IDM stimulation, but not to TCDD, was maintained in AhR-null fibroblasts. CYP1B1 expression, unlike TCDD induction, was stimulated by IDM in only about half the cells. CYP1B1 expression partially overlapped with PPARgamma expression, which was also inversely related in clonal sub-lines. CYP1B1 expression may, therefore, represent an early stage of differentiation that requires factors associated with DNA synthesis to subsequently generate PPARgamma1.

Mechanisms of erythropoietin-mediated cardioprotection during ischemia-reperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling.

Langendorff-perfused rat hearts treated with EPO exhibited significantly improved postischemic recovery of left ventricular developed pressure (LVDP) and reduced infarct size compared with control hearts. Perfusion with the mitogen/extracellular signal-regulated kinase (MEK) inhibitor U0126 just before and concomitant with EPO treatment abolished EPO-induced phosphorylation of the MEK substrate extracellular signal-regulated kinase (ERK) but had no effect of EPO-mediated cardioprotection. EPO treatment of the perfused hearts induced translocation of protein kinase C (PKC) epsilon isoform to the membrane fraction of the hearts and the protective effect of EPO was significantly inhibited by the PKC catalytic inhibitor chelerythrine added before and concomitant with EPO. These data demonstrate that EPO-mediated activation of the PKC signaling pathway before or during ischemia is required for the cardioprotective effect of EPO during ischemia-reperfusion injury. Perfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin just before and concomitant with EPO treatment attenuated EPO-induced phosphorylation of the PI3K substrate Akt but had no effect on EPO-mediated cardioprotection. However, when wortmannin was added during EPO treatment and continued during reperfusion, EPO-mediated cardioprotection was significantly inhibited. We also show that postischemia EPO treatment at the onset of reperfusion significantly improved recovery of LVDP and reduced infarct size. Postischemia cardioprotection by EPO required the PI3K pathway but was not affected by inhibition of PKC at the time of EPO treatment.

Identification of novel TCDD-regulated genes by microarray analysis.

TCDD exposure of multipotential C3H10T1/2 fibroblasts for 72 h altered the expression of over 1000 genes, including coordinated changes across large functionally similar gene clusters. TCDD coordinately induced 23 cell cycle-related genes similar to epidermal growth factor (EGF)-induced levels but without any affect on the major mitogenic signaling pathway (extracellular signal-regulated kinase, ERK). TCDD treatment also decreased glycolytic and ribosomal clusters. Most of these TCDD-induced changes were attenuated by the presence of EGF or an adipogenic stimulus, each added during the final 24 h. TCDD prevented 10% of EGF-induced gene responses and 40% of adipogenic responses. Over 100 other genes responded to TCDD during adipogenesis. This group of responses included complete suppression of three proliferins and stimulations of several cytokine receptors. Despite these varied secondary effects of TCDD, direct AhR activation measured by integrated AhR-responsive luciferase reporters was similar under quiescent, EGF-stimulated or adipogenic conditions. Only 23 genes were similarly induced by TCDD regardless of conditions and 10 were suppressed. These 23 genes include: 4 genes previously recognized to contain AhR response elements (cytochrome P450 (CYP) 1B1, CYP1A1, NAD(P)H quinone reductase 1 (NQO1), and aldehyde dehydrogenase 3A1); two novel oxidative genes (alcohol dehydrogenase 3 and superoxide dismutase 3); and glypican 1, a plasma membrane proteoglycan that affects cell signaling. Further experiments demonstrated that TCDD maximally induced NQO1, glypican 1 and alcohol dehydrogenase 3 by 6 h. Glypican 1 activates the actions of many growth factors and therefore may contribute to secondary effects on gene expression.

Ephedrine plus caffeine causes age-dependent cardiovascular responses in Fischer 344 rats.

Human consumption of ephedrine and caffeine in dietary supplements has been associated with a number of adverse effects including changes in the ECG, myocardial infarction, hyperthermia, and, in rare instances, death. The purpose of this study was to investigate the potential mechanisms associated with the cardiotoxicity of combined ephedrine and caffeine ingestion. Seven- and fourteen-week-old Fischer 344 rats treated with ephedrine in combination with caffeine exhibited increases in heart rate (HR), temperature, and corrected QT interval. Of the 14-wk-old rats treated with 25 mg/kg ephedrine plus 30 mg/kg caffeine, 57% died within 3-5 h of treatment, whereas none of the similarly treated 7-wk-old rats nor any of the rats treated with vehicle died. One hour after treatment with this dose of ephedrine plus caffeine, 14-wk-old rats exhibited a larger increase in HR (as % increase over baseline) than 7-wk-old rats. Furthermore, the 14-wk-old rats that died had a higher HR and temperature than the 14-wk-old rats that lived. Histopathological studies suggested interstitial hemorrhage and myofiber necrosis in the 14-wk-old rats treated with the highest concentration of ephedrine and caffeine. This study showed enhanced susceptibility to ephedrine plus caffeine in 14-wk-old rats compared with 7-wk-old rats. The greater mortality in the 14-wk-old rats was associated with increases in body temperature, HR, and myocardial necrosis.

TCDD administration after the pro-adipogenic differentiation stimulus inhibits PPARgamma through a MEK-dependent process but less effectively suppresses adipogenesis.

Hormone (IDMB)-induced adipogenesis in C3H10T1/2 cells is suppressed by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon receptor (AhR). We have previously reported that TCDD addition 48 h before the hormonal stimulation of IDMB suppresses a key mediator of adipogenesis, the peroxisome proliferator-activated receptor (PPARgamma), by a MEK/ERK dependent mechanism. Here we add to previous evidence that this synergism functions after IDMB addition but before increased PPARgamma1 transcription. Suppression remains effective and MEK/ERK dependent when TCDD is added 6-12 h after IDMB addition but not when delayed to 16-24 h, thus preceding the rise in PPARgamma mRNA. TCDD suppression of the number of committed adipocytes and of triglyceride formation is less effective with the delayed addition. TCDD therefore does not directly suppress the expression of the key mediator PPARgamma1. An alternative mediation of adipocyte commitment is apparently less sensitive to the 6-12 h of delayed TCDD addition. TCDD suppression potencies (EC(50) = 50 pM) match the potencies for stimulation of CYP1B1 protein and AhR-sensitive reporters. The AhR antagonist 3'-methoxy-4'-nitroflavone (3-MNF) inhibited both TCDD-mediated CYP1B1 induction and inhibition of PPARgamma protein expression. This antagonism was only effective when 3-MNF was present in the 24-h period after IDMB addition. TCDD activation of AhR in conjunction with MEK/ERK therefore generates PPARgamma1 suppression activity before the increase of PPARgamma1 synthesis. The potency and inhibition data are consistent with induction of one or more gene products that sustain suppression through the extended period of PPARgamma1 transcription.

Effects of inorganic HgCl2 on adipogenesis.

Mercury is a common pollutant that alters glucose metabolism in adipocytes; however, the effect of HgCl2 on differentiating adipocytes and their subsequent metabolic function is not well understood. Two adipocyte models, the 3T3-L1 and C3H10T1/2 (10T1/2) cell lines, were differentiated in the presence of HgCl2. To assess the amount of differentiation in a population, markers of differentiation (i.e., PPARgamma and GLUT 4 expression and lipid accumulation) and functions of adipocytes (i.e., glucose transport and insulin-induced glucose transport) were measured. HgCl2 exposure significantly decreased the number of phenotypic adipocytes and PPARgamma expression in both 3T3-L1 and 10T1/2 cells without effects on cell viability. GLUT 4 was significantly reduced by HgCl2 treatment in 10T1/2 but not 3T3-L1 cells. Exposure to HgCl2 during differentiation increased basal glucose uptake in a dose-dependent manner (up to 2.5-fold) and decreased insulin-induced glucose uptake in 3T3-L1 adipocytes. In contrast, HgCl2 had little effect on basal or insulin-induced glucose uptake in 10T1/2 cells, possibly due to their lower insulin responsiveness. We examined the effect of HgCl2 exposure on signaling event involved in differentiation of adipocytes and cellular stress, namely, the phosphorylation of ERK1/2 and JNK, respectively. HgCl2 exposure had no effect on ERK1/2 phosphorylation in either cell line, increased JNK phosphorylation in the 10T1/2, and had no effect on JNK phosphorylation in 3T3-L1 cells. These data indicate HgCl2 exposure can inhibit the differentiation of fibroblasts into adipocytes as well as influence signaling events and the subsequent metabolic activity of differentiated adipocytes.

AhR- and ERK-dependent pathways function synergistically to mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin suppression of peroxisome proliferator-activated receptor-gamma1 expression and subsequent adipocyte differentiation.

Activation of the aryl-hydrocarbon receptor (AhR) by pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) blocks hormone (IDM/BRL)-induced adipocyte differentiation of C3H10T1/2 cells in proportion to the suppression of the elevation of the key mediator, peroxisome proliferator-activated receptor (PPARgamma1). Inhibition of MEK-induced ERK phosphorylation had no effect on adipogenesis but prevented this TCDD suppression. Initiation of MEK inhibition up to 6 h after IDM/BRL stimulation in combination with serum addition completely reversed the TCDD-mediated suppression but declined to ineffectiveness when delayed to 24 h after stimulation. This period occurs well after the decline of serum-induced ERK activation, at a time when ERK phosphorylation is low, and prior to the onset of IDM/BRL-stimulated PPARgamma1 expression. This temporal separation of ERK activation from the affected PPARgamma1 expression suggests that ERK does not act directly on either PPARgamma1 transcription or receptor function. Thus, ERK activation and TCDD/AhR stimulation work synergistically to inhibit adipocyte differentiation. Nonrenewal of serum at the time of IDM/BRL addition removed most of the ERK activation and also the TCDD-mediated suppressions of PPARgamma1 expression and adipocyte differentiation. Transfection of a vector expressing constitutively active MEK1 generated a constant, high level of phosphorylated ERK comparable to the peak serum-induced level and fully restored TCDD suppression without a TCDD-mediated effect on ERK phosphorylation. We conclude that low levels of activated MEK and ERK cooperate with AhR-induced factor(s) to generate a suppressor that prevents PPARgamma1 transcription and then differentiation.