Cytotoxic, genotoxic, and toxicogenomic effects of dihydroxyacetone in human primary keratinocytes.

PURPOSE: Dihydroxyacetone (DHA) is the only ingredient approved by the U.S. FDA as a colour additive in sunless tanning (self-tanning) products. Consumer sunless tanning products available for retail purchase contain 1-15% DHA. Although originally thought to only interact with the stratum corneum, more recent research has shown that DHA penetrates beyond the stratum corneum to living keratinocytes indicating a possible route of exposure in the epidermis. MATERIALS AND METHODS: Normal Human Epidermal Keratinocytes (NHEK) were used to determine any potential in vitro toxicological effects of DHA in the epidermis. NHEK cells exposed to DHA concentrations up to 0.90% (100 mM) in dosing media were evaluated for viability, genotoxicity (Comet Assay), and gene expression changes by microarray analysis. RESULTS: Cell viability significantly decreased ∼50% after 3-h exposure to 50 and 100 mM DHA. DNA damage was only found to be significantly increased in cells exposed to cytotoxic DHA concentrations. A subtoxic dose of DHA induced significant gene expression changes. Particularly, expression of cyclin B1, CDK1, and six other genes associated with the G2/M cell cycle checkpoint was significantly decreased which correlates well with a G2/M block reported in the existing literature. Advanced Glycation End Product (AGE) formation significantly increased after 24 h of DHA exposure at and above 10 mM. In summary, these data show that DHA is cytotoxic above 25 mM in primary keratinocytes. Genotoxicity was detected only at cytotoxic concentrations, likely indicative of non-biologically relevant DNA damage, while subtoxic doses induce gene expression changes and glycation. CONCLUSION: DHA treatment had a significant and negative effect on primary keratinocytes consistent with in vitro cultured cell outcomes; however, more information is needed to draw conclusions about the biological effect of DHA in human skin.

Regulation of Caenorhabditis elegans vitellogenesis by DAF-2/IIS through separable transcriptional and posttranscriptional mechanisms.

BACKGROUND: Evolutionary theories of aging propose that longevity evolves as a competition between reproduction and somatic maintenance for a finite pool of resources. Reproduction is thought to shorten lifespan by depleting resources from processes promoting somatic maintenance. Maternal yolk production, vitellogenesis, represents a significant maternal cost for reproduction and is suppressed under genetic and environmental conditions that extend lifespan. However, little is known about the pathways regulating vitellogenesis in response to prolongevity cues. RESULTS: In order to identify mechanisms that suppress vitellogenesis under prolongevity conditions, we studied factors regulating vitellogenesis in C. elegans nematodes. In C. elegans, vitellogenesis is depressed in the absence of insulin-like signaling (IIS). We found that the C. elegans daf-2/IIS pathway regulates vitellogenesis through two mechanisms. vit-2 transcript levels in daf-2 mutants were indirectly regulated through a germline-dependent signal, and could be rescued by introduction of daf-2(+) sperm. However, yolk protein (YP) levels in daf-2 mutants were also regulated by germline-independent posttranscriptional mechanisms. CONCLUSIONS: C. elegans vitellogenesis is regulated transcriptionally and posttranscriptionally in response to environmental and reproductive cues. The daf-2 pathway suppressed vitellogenesis through transcriptional mechanisms reflecting reproductive phenotypes, as well as distinct posttranscriptional mechanisms. This study reveals that pleiotropic effects of IIS pathway mutations can converge on a common downstream target, vitellogenesis, as a mechanism to modulate longevity.

Biomarkers of Inflammation and Oxidative Stress among Adult Former Smoker, Current E-Cigarette Users-Results from Wave 1 PATH Study.

BACKGROUND: Former smokers who currently use e-cigarettes have lower concentrations of biomarkers of tobacco toxicant exposure than current smokers. It is unclear whether tobacco toxicant exposure reductions may lead to health risk reductions. METHODS: We compared inflammatory biomarkers (high-sensitivity C-reactive protein, IL6, fibrinogen, soluble intercellular adhesion molecule-1) and an oxidative stress marker (F2-isoprostane) among 3,712 adult participants in Wave 1 (2013-2014) of the Population Assessment of Tobacco and Health Study by tobacco user groups: dual users of cigarettes and e-cigarettes; former smokers who currently use e-cigarettes-only; current cigarette-only smokers; former smokers who do not currently use any tobacco; and never tobacco users. We calculated geometric means (GM) and estimated adjusted GM ratios (GMR). RESULTS: Dual users experienced greater concentration of F2-isoprostane than current cigarette-only smokers [GMR 1.09 (95% confidence interval, CI, 1.03-1.15)]. Biomarkers were similar between former smokers who currently use e-cigarettes and both former smokers who do not use any tobacco and never tobacco users, but among these groups most biomarkers were lower than those of current cigarette-only smokers. The concentration of F2-isoprostane decreased by time since smoking cessation among both exclusive e-cigarette users (P trend = 0.03) and former smokers who do not currently use any tobacco (P trend = 0.0001). CONCLUSIONS: Dual users have greater concentration of F2-isoprostane than smokers. Exclusive e-cigarette users have biomarker concentrations that are similar to those of former smokers who do not currently use tobacco, and lower than those of exclusive cigarette smokers. IMPACT: This study contributes to an understanding of the health effects of e-cigarettes.

Diglycolic acid induces HepG2/C3A liver cell toxicity in vitro.

Carboxymethyl starches are added to food products for thickening or tablet binding/filling purposes. Although they lack toxicity, their synthesis creates the chemical byproduct diglycolic acid (DGA), which is difficult to eliminate and whose toxicity is in question. A rare case of an accidental direct exposure to extremely high concentrations of DGA in a person revealed that DGA has the potential to be toxic to several organs, with the kidneys and liver being the most affected organs. Given that DGA is present in our food supply as a chemical byproduct of carboxymethyl starch food additives, we sought to perform in vitro testing of its potential hepatotoxicity to help complement a recent in vivo rat acute dose-response study that also tested for the potential hepatotoxic effects of daily DGA ingestion by oral gavage over a period of 28 days. Using the HepG2/C3A cellular in vitro model, we tested how escalating doses of DGA exposure over 24 h could induce hepatotoxicity. Both in vitro and in vivo testing systems revealed that DGA is indeed a hepatotoxin once a certain exposure threshold is reached. The concordance of these models highlights the utility of in vitro testing to support and help predict in vivo findings.

Comparison of diglycolic acid exposure to human proximal tubule cells in vitro and rat kidneys in vivo.

Diglycolic acid (DGA) is present in trace amounts in our food supply and is classified as an indirect food additive linked with the primary GRAS food additive carboxymethyl cellulose (CMC). Carboxymethyl starches are used as a filler/binder excipient in dietary supplement tablets and a thickening ingredient in many other processed foods. We sought to utilize the human proximal tubule HK-2 cell line as an in vitro cellular model system to evaluate its acute nephrotoxicity of DGA. We found that DGA was indeed toxic to HK-2 cells in all in vitro assays in our study, including a highly sensitive Luminex assay that measures levels of an in vitro biomarker of kidney-specific toxicity, Kidney Injury Molecule 1 (KIM-1). Interestingly, in vitro KIM-1 levels also correlated with in vivo KIM-1 levels in urine collected from rats treated with DGA by daily oral gavage. The use of in vitro and in vivo models towards understanding the effectiveness of an established in vitro system to predict in vivo outcomes would be particularly useful in rapidly screening compounds that are suspected to be unsafe to consumers. The merit of the HK-2 cell model in predicting human toxicity and accelerating the process of food toxicant screening would be especially important for regulatory purposes. Overall, our study not only revealed the value of HK-2 in vitro cell model for nephrotoxicity evaluation, but also uncovered some of the mechanistic aspects of the human proximal tubule injury that DGA may cause.

Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extracts.

Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans-Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of 0.30 microm/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 microm/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes.

The Krüppel-like factor KLF15 regulates the insulin-sensitive glucose transporter GLUT4.

Resistance to the stimulatory effects of insulin on glucose utilization is a key feature of type 2 diabetes, obesity, and the metabolic syndrome. Recent studies suggest that insulin resistance is primarily caused by a defect in glucose transport. GLUT4 is the main insulin-responsive glucose transporter and is expressed predominantly in muscle and adipose tissues. Whereas GLUT4 has been shown to play a critical role in maintaining systemic glucose homeostasis, the mechanisms regulating its expression are incompletely understood. We have cloned the murine homologue of KLF15, a member of the Krüppel-like family of transcription factors. KLF15 is highly expressed in adipocytes and myocytes in vivo and is induced when 3T3-L1 preadipocytes are differentiated into adipocytes. Overexpression of KLF15 in adipose and muscle cell lines potently induces GLUT4 expression. This effect is specific to KLF15 as overexpression of two other Krüppel-like factors, KLF2/LKLF and KLF4/GKLF, did not induce GLUT4 expression. Both basal (3.3-fold, p < 0.001) and insulin-stimulated (2.4-fold, p < 0.00001) glucose uptake are increased in KLF15-overexpressing adipocytes. In co-transfection assays, KLF15 and MEF2A, a known activator of GLUT4, synergistically activates the GLUT4 promoter. Promoter deletion and mutational analyses provide evidence that this activity requires an intact KLF15-binding site proximal to the MEF2A site. Finally, co-immunoprecipitation assays show that KLF15 specifically interacts with MEF2A. These studies indicate that KLF15 is an important regulator of GLUT4 in both adipose and muscle tissues.

Transforming growth factor-beta1 inhibition of vascular smooth muscle cell activation is mediated via Smad3.

Activation of vascular smooth muscle cells (VSMCs) by proinflammatory cytokines is a key feature of atherosclerotic lesion formation. Transforming growth factor (TGF)-beta1 is a pleiotropic growth factor that can modulate the inflammatory response in diverse cell types including VSMCs. However, the mechanisms by which TGF-beta1 is able to mediate these effects remains incompletely understood. We demonstrate here that the ability of TGF-beta1 to inhibit markers of VSMC activation, inducible nitric-oxide synthase (iNOS) and interleukin (IL)-6, is mediated through its downstream effector Smad3. In reporter gene transfection studies, we found that among a panel of Smads, Smad3 could inhibit iNOS induction in an analogous manner as exogenous TGF-beta1. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous iNOS and IL-6. Conversely, TGF-beta1 inhibition of cytokine-mediated induction of iNOS and IL-6 expression was completely blocked in Smad3-deficient VSMCs. Previous studies demonstrate that CCAAT/enhancer-binding protein (C/EBP) and NF-kappaB sites are critical for cytokine induction of both the iNOS and IL-6 promoters. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on C/EBP DNA-protein binding and activity. Smad3 mediates this effect in part by inhibiting C/EBP-beta and C/EBP-delta through distinct mechanisms. Furthermore, we find that Smad3 prevents the cooperative induction of the iNOS promoter by C/EBP and NF-kappaB. These data demonstrate that Smad3 plays an essential role in mediating TGF-beta1 anti-inflammatory response in VSMCs.