Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

Ablation of sphingosine kinase 2 suppresses fatty liver-associated hepatocellular carcinoma via downregulation of ceramide transfer protein.

Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancer, the third leading cause of cancer-associated death worldwide. With the increasing prevalence of metabolic conditions, non-alcoholic fatty liver disease (NAFLD) is emerging as the fastest-growing HCC risk factor, and it imposes an additional layer of difficulty in HCC management. Dysregulated hepatic lipids are generally believed to constitute a deleterious environment cultivating the development of NAFLD-associated HCC. However, exactly which lipids or lipid regulators drive this process remains elusive. We report herein that sphingosine kinase 2 (SphK2), a key sphingolipid metabolic enzyme, plays a critical role in NAFLD-associated HCC. Ablation of Sphk2 suppressed HCC development in NAFLD livers via inhibition of hepatocyte proliferation both in vivo and in vitro. Mechanistically, SphK2 deficiency led to downregulation of ceramide transfer protein (CERT) that, in turn, decreased the ratio of pro-cancer sphingomyelin (SM) to anti-cancer ceramide. Overexpression of CERT restored hepatocyte proliferation, colony growth and cell cycle progression. In conclusion, the current study demonstrates that SphK2 is an essential lipid regulator in NAFLD-associated HCC, providing experimental evidence to support clinical trials of SphK2 inhibitors as systemic therapies against HCC.

Ceramide Transfer Protein (CERT): An Overlooked Molecular Player in Cancer.

Sphingolipids are a class of essential lipids implicated in constructing cellular membranes and regulating nearly all cellular functions. Sphingolipid metabolic network is centered with the ceramide-sphingomyelin axis. Ceramide is well-recognized as a pro-apoptotic signal; while sphingomyelin, as the most abundant type of sphingolipids, is required for cell growth. Therefore, the balance between these two sphingolipids can be critical for cancer cell survival and functioning. Ceramide transfer protein (CERT) dictates the ratio of ceramide to sphingomyelin within the cell. It is the only lipid transfer protein that specifically delivers ceramide from the endoplasmic reticulum to the Golgi apparatus, where ceramide serves as the substrate for sphingomyelin synthesis. In the past two decades, an increasing body of evidence has suggested a critical role of CERT in cancer, but much more intensive efforts are required to draw a definite conclusion. Herein, we review all research findings of CERT, focusing on its molecular structure, cellular functions and implications in cancer. This comprehensive review of CERT will help to better understand the molecular mechanism of cancer and inspire to identify novel druggable targets.

Ceramide Regulates Anti-Tumor Mechanisms of Erianin in Androgen-Sensitive and Castration-Resistant Prostate Cancers.

Prostate cancer is the second most prevalent malignancy worldwide. In the early stages, the development of prostate cancer is dependent on androgens. Over time with androgen deprivation therapy, 20% of prostate cancers progress to a castration-resistant form. Novel treatments for prostate cancers are still urgently needed. Erianin is a plant-derived bibenzyl compound. We report herein that erianin exhibits anti-tumor effects in androgen-sensitive and castration-resistant prostate cancer cells through different mechanisms. Erianin induces endoplasmic reticulum stress-associated apoptosis in androgen-sensitive prostate cancer cells. It also triggers pro-survival autophagic responses, as inhibition of autophagy predisposes to apoptosis. In contrast, erianin fails to induce apoptosis in castration-resistant prostate cancer cells. Instead, it results in cell cycle arrest at the M phase. Mechanistically, C16 ceramide dictates differential responses of androgen-sensitive and castration-resistant prostate cancer cells to erianin. Erianin elevates C16 ceramide level in androgen-sensitive but not castration-resistant prostate cancer cells. Overexpression of ceramide synthase 5 that specifically produces C16 ceramide enables erianin to induce apoptosis in castration-resistant prostate cancer cells. Our study provides both experimental evidence and mechanistic data showing that erianin is a potential treatment option for prostate cancers.

PTU, a novel ureido-fatty acid, inhibits MDA-MB-231 cell invasion and dissemination by modulating Wnt5a secretion and cytoskeletal signaling.

Migration and invasion promote tumor cell metastasis, which is the leading cause of cancer death. At present there are no effective treatments. Epidemiological studies have suggested that ω-3 polyunsaturated fatty acids (PUFA) may decrease cancer aggressiveness. In recent studies epoxide metabolites of ω-3 PUFA exhibited anti-cancer activity, although increased in vivo stability is required to develop useful drugs. Here we synthesized novel stabilized ureido-fatty acid ω-3 epoxide isosteres and found that one analogue - p-tolyl-ureidopalmitic acid (PTU) - inhibited migration and invasion by MDA-MB-231 breast cancer cells in vitro and in vivo in xenografted nu/nu mice. From proteomics analysis of PTU-treated cells major regulated pathways were linked to the actin cytoskeleton and actin-based motility. The principal finding was that PTU impaired the formation of actin protrusions by decreasing the secretion of Wnt5a, which dysregulated the Wnt/planar cell polarity (PCP) pathway and actin cytoskeletal dynamics. Exogenous Wnt5a restored invasion and Wnt/PCP signalling in PTU-treated cells. PTU is the prototype of a novel class of agents that selectively dysregulate the Wnt/PCP pathway by inhibiting Wnt5a secretion and actin dynamics to impair MDA-MB-231 cell migration and invasion.

Simulations and Experimental Verifications of an Algorithm for Radiation Source Mapping and Navigational Path Generation.

ABSTRACT: Accurate and efficient mapping and localization of both ionizing and non-ionizing radiation sources are important across many different fields. As such, a versatile mapping and navigational path generation algorithm, which can be applied to any point source measurements that follow an inverse-square characteristic, was developed using non-linear least squares methods. Forty thousand simulations were performed on the algorithm, which located sources successfully in a 10 m × 10 m × 10 m three-dimensional space with a success rate of over 80% across different noise functions, given a proportional constant of 10 to 1,000. The algorithm was also verified experimentally with small-scale radioactive decontamination of a 70 cm × 70 cm surface and localization of a lost Wi-Fi router in a 70 m × 70 m open field. One hundred twenty-one measurements were taken from each experiment, which were then fed into the algorithm for navigation. For the radioactive 137Cs source, the estimated locations were within 7 cm × 7 cm of the answer in 79.3% of the scenarios, while the Wi-Fi router was located to within 7 m × 7 m in 57.9% of the tests. In general, the method requires much less information and data than a geographically comprehensive survey and thus shows a lot of potential for practical applications, such as lost source retrieval with unmanned aerial vehicles, small-scale decontamination, mapping undocumented Wi-Fi routers or radio towers, and radiation simulation with radio signals. Different failure modes, desirable features, and potential improvements were also identified but remain as future work.

Integrated Glycoproteomics Identifies a Role of N-Glycosylation and Galectin-1 on Myogenesis and Muscle Development.

Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases, suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development, but our molecular understanding of the precise glycans, catalytic enzymes, and lectins involved remains only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins, most notably the upregulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation, suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the upregulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.

Radiation Mapping for an Unmanned Aerial Vehicle: Development and Simulated Testing of Algorithms for Source Mapping and Navigation Path Generation.

ABSTRACT: Image reconstruction algorithms were developed for radiation source mapping and used for generating the search path of a moving radiation detector, such as one onboard an unmanned aerial vehicle. Simulations consisted of first assuming radioactive sources of varying complexity and estimating the radiation fields that would then be produced by that source distribution. Next, the "measurements" that would result from a pair of adjacent spatial locations were computed. A crude estimate of the source distribution likely to have produced such "measurements" was reconstructed based upon the limited measurements. Location of the next "measurement" was then determined as halfway between the location of the estimated source and the current "measurement." With each additional sample, improved source distribution reconstructions were made and used to inform the immediate direction of detector motion. Source reconstruction or mapping was formulated as an inverse problem solved with either maximum a posteriori or least squares (LS) regression deconvolution methods. Different amounts of noise were added to the simulated "measurements," allowing evaluation of the methods' performances as functions of signal-to-noise ratio of the measured map. As expected, methods that promote sparsity were better suited in reconstructing point sources. Reliable prior information of the source distribution also improved the reconstruction results, especially with distributed sources. With a non-negative least square algorithm and the suggested paths it generated, location of sources was successfully estimated to an accuracy of 0.014 m within nine iterations in a single-source scenario and 12 iterations in a two-source scenario, given a 10% error on the integrated counts and a Poisson distribution of the noise associated with the measured counts.

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2.

Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.

The Effects of Radiation and Emitted Light Transport on the Positional Response of 11 cm × 42.5 cm × 5.5 cm NaI(Tl) Detectors.

Experiments were performed with 30 11 cm × 42.5 cm × 5.5 cm NaI(Tl) detectors to better understand their positional response. Spectra were collected using 0.02 to 0.15 MBq point sources of Am, Cs, Co, and Ba positioned on lines parallel and perpendicular to the long axis of the crystal along both the narrow and wide detector faces as well as at different distances from them. A greater density of positions was sampled at the ends of the detector, and repeated measurements were made to examine potential gain drifts during the experiment. Spectroscopic peak counts, spectroscopic pulse heights, and net counts were analyzed. Empirical equations were fit to the aforementioned data for each specific source energy as a function of source position. In addition, a Monte Carlo radiation transport code was used to simulate the expected positionally variable response based solely upon radiation absorption. The simulated radiation transport efficiency functions were compared to the experimental data. The effects of the geometric radiation efficiency, the attenuation and scattering of emitted light within the scintillation crystal, and combined effects such as nonuniformity of the photomultiplier tube, photocathode response, and crystal irregularities were then distinguished. Functions describing each effect were derived. The results suggest potential new corrections to data obtained with large scintillation detectors as well as a novel approach to partial positional gamma-ray detection with minimal collimation, given that the energy resolution is within reason for particular photopeaks.

The Interaction of the Metallo-Glycopeptide Anti-Tumour Drug Bleomycin with DNA.

The cancer chemotherapeutic drug, bleomycin, is clinically used to treat several neoplasms including testicular and ovarian cancers. Bleomycin is a metallo-glycopeptide antibiotic that requires a transition metal ion, usually Fe(II), for activity. In this review, the properties of bleomycin are examined, especially the interaction of bleomycin with DNA. A Fe(II)-bleomycin complex is capable of DNA cleavage and this process is thought to be the major determinant for the cytotoxicity of bleomycin. The DNA sequence specificity of bleomycin cleavage is found to at 5′-GT* and 5′-GC* dinucleotides (where * indicates the cleaved nucleotide). Using next-generation DNA sequencing, over 200 million double-strand breaks were analysed, and an expanded bleomycin sequence specificity was found to be 5′-RTGT*AY (where R is G or A and Y is T or C) in cellular DNA and 5′-TGT*AT in purified DNA. The different environment of cellular DNA compared to purified DNA was proposed to be responsible for the difference. A number of bleomycin analogues have been examined and their interaction with DNA is also discussed. In particular, the production of bleomycin analogues via genetic manipulation of the modular non-ribosomal peptide synthetases and polyketide synthases in the bleomycin gene cluster is reviewed. The prospects for the synthesis of bleomycin analogues with increased effectiveness as cancer chemotherapeutic agents is also explored.

The mitochondrial DNA sequence specificity of the anti-tumour drug bleomycin using end-labeled DNA and capillary electrophoresis and a comparison with genome-wide DNA sequencing.

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was investigated in two human mitochondrial DNA sequences. Bleomycin was found to cleave preferentially at 5'-TGT*A-3' DNA sequences (where * is the cleavage site). The bleomycin analysis using capillary electrophoresis with laser-induced fluorescence was determined on both DNA strands and each strand was independently fluorescently labelled at the 3'- and 5'-ends. There was a high level of correlation between the intensity of bleomycin cleavage sites analysed by 3'- and 5'-end labelling. This is the first occasion that a comprehensive comparison has been made between these two end-labelling procedures to quantify cleavage by a DNA damaging agent and to investigate end-label bias. A comparison was also made between the bleomycin DNA sequence specificity obtained from genome-wide next-generation sequencing with that obtained from purified plasmid DNA sequences. This was accomplished by cloning sections of human mitochondrial DNA and comparing these identical mitochondrial DNA in the human mitochondrial genome. At individual sites, there was a very low level of correlation between bleomycin cleavage in plasmid sequencing and genome-wide sequencing. However, the overall bleomycin DNA sequence specificity was very similar in the two environments, namely 5'-TGT*A-3'.

Intravenous thiamylal and local anesthetic infiltration for pediatric facial repair procedures performed in emergency departments.

Thiamylal is widely used for procedural sedation in emergency departments (ED); however, there are limited safety data for doses of thiamylal > 5 mg/kg in children. We investigated whether intravenous thiamylal in combination with local anesthetics is safe and effective for pediatric procedural sedation in the ED and to identify the association between increasing doses thiamylal and adverse events. Between July 2004 and June 2008, 227 children who underwent procedural sedation met the inclusion criteria, including 105 males (46.3%) and 122 females (53.7%). Facial laceration was the most common indication for procedural sedation. All children received an intravenous injection of thiamylal, with a loading dose of 5 mg/kg. Eighty-one children (35.7%) received a supplemental dose of 2.5 mg/kg thiamylal because of inadequate sedation. Of these, 27 (11.9%) received a second supplemental dose of 2.5 mg/kg because of inadequate sedation. Sixty-six patients (29.1%) experienced 75 mild and self-resolving adverse events, and most of which (15/75; 20%) were drowsiness. Four (1.8%) patients experienced oxygen saturation below 96%, which was related to the supplemental dose of thiamylal (p = 0.002). No children suffered from any lasting or potentially serious complications. Our results indicate that intravenous thiamylal in combination with local anesthetic infiltration is a well tolerated for therapeutic procedures in the ED. Thiamylal offers rapid onset of sedation without compromising the patient's cardiorespiratory function during pediatric procedural sedation.

Glucosamine sulfate inhibits TNF-alpha and IFN-gamma-induced production of ICAM-1 in human retinal pigment epithelial cells in vitro.

PURPOSE: Glucosamine sulfate (GS) is a naturally occurring sugar that possesses some immunosuppressive effects in vitro and in vivo, but its mechanism is unknown. We investigated whether GS could modulate the proinflammatory cytokine-induced expression of the gene for intercellular adhesion molecule (ICAM)-1, an inflammatory protein in human retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used as a model to determine the effects of GS on the expression of the ICAM-1 gene upregulated by TNF-alpha or IFN-gamma, by Western blot analysis and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The activation and nuclear translocation of the nuclear factors NF-kappaB and STAT1 were evaluated by immunocytochemistry, Western blot analysis, and electrophoretic mobility shift assay (EMSA). RESULTS: Both TNF-alpha and IFN-gamma increased the expression of ICAM-1 at the mRNA and protein levels in a time- and dose-dependent manner in ARPE-19 cells. GS effectively downregulated the TNF-alpha- or IFN-gamma-induced expression of ICAM-1 in the protein and mRNA level in a dose-dependent manner. GS further inhibited the nuclear translocation of p65 proteins in TNF-alpha and phosphorylated STAT1 in IFN-gamma-stimulated ARPE-19 cells. CONCLUSIONS: GS inhibits the expression of the ICAM-1 gene in ARPE-19 cell stimulated with TNF-alpha or IFN-gamma through blockade of NF-kappaB subunit p65 and nuclear translocation of STAT1. This study has demonstrated a potentially important property of GS in reducing ICAM-1 mediated inflammatory mechanisms in the eye.

Human telomerase inhibition and cytotoxicity of regioisomeric disubstituted amidoanthraquinones and aminoanthraquinones.

Telomerase is an attractive target for the rational design of new anticancer drugs due to its central role in the control of cellular proliferation. A number of 1,4-disubstituted amidoanthraquinones and 1,5-disubstituted aminoanthraquinones that are related to mitoxantrone and ametantrone have previously been prepared. The present study details the effects on human telomerase of these new classes of 1,4- and 1,5-difunctionalized tricyclic anthraquinone compounds. We have used cytotoxicity assay, reporter SEAP assay to monitor the hTERT expression, and TRAP-G4 assay to measure the relative activity of these compounds, and have examined how the attached substituents affect their ability to influence telomerase. Cytotoxicity levels in human tumor cell lines were at comparable levels for several compounds. Structural and activity relationships indicated that the position of disubstituent side chains is important for its inhibitory effect. Moreover, a primary amine or tertiary amine on the substitution group appears to be required for the telomerase inhibitory effect. There is no significant correlation between telomerase activity and cytotoxicity. These symmetrical disubstituted anthraquinones may represent useful leads for the development of human telomerase inhibitors as potential anticancer agents, and the exact mode of intercalative binding is dictated by the positional placement of substituent side chains for effective telomerase inhibition.

Retinoic acid induces VEGF gene expression in human retinal pigment epithelial cells (ARPE-19).

PURPOSE: The aim of this study was to evaluate the expression of vascular endothelial growth factor (VEGF) in response to retinoic acid (RA) in human retinal pigment epithelial cells. METHODS: Expression of VEGF in human ARPE-19 cells was determined by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). mRNA stability was assessed after the administration of actinomycin D. The induction of the VEGF gene by various RAs was also determined by semiquantitative RT-PCR. RESULTS: All-trans retinoic acid (atRA) time-dependently increased VEGF mRNA levels. The effect of atRA was dose-dependent in a range between 10(-7) M and 10(-6) M. Treatment with actinomycin D revealed that atRA induces the VEGF gene at the transcriptional level. Of the various RAs tested, atRA was the most potent inducer of the VEGF gene. CONCLUSIONS: We demonstrated that atRA stimulates the induction of the VEGF gene in ARPE-19 cells, suggesting a novel pathway for the development of age-related macular degeneration.

Small-molecule anthracene-induced cytotoxicity and induction of apoptosis through generation of reactive oxygen species.

A series of anthracene derivatives have been synthesized, and their potential individual cytotoxicity was evaluated using Jurkat T cells and peripheral blood mononuclear cells (PBMCs) in vitro. These compounds, except for 2l, showed less cytotoxicity in PBMCs than mitoxantrone. We also analyzed the antiproliferative activity of these derivatives using the annexin V/propidium iodide assay. These synthetic compounds induced apoptosis, thus leading to antitumor effects. Compounds 2b, 2e, 2f, 2g, 2h, 2i, 2j, and mitoxantrone produced dose-dependent cytotoxicity, while the antiproliferative activity of the anthracene pharmacophore was retained in Jurkat T cells base on the detection of DNA degradation and membrane unpacking. These clearly indicate a correlation between cytotoxicity and antitumor activity. Unlike mitoxantrone, cytotoxic properties were observed, as documented by the reactivity of these novel compounds against Jurkat T cells and PBMCs as normal cells, respectively. Various concentrations of 2b, 2e, 2f, 2g, 2h, 2i, and 2j preparations also inhibited Jurkat T cell proliferation and induced apoptosis of Jurkat T cells, potentially confirmed through the detection of DNA degradation and membrane unpacking. In the present report we also investigated the antiinflammatory activity against phorbol-12-myristate-13-acetate induced superoxide anion production, a marker for an inflammatory mediator produced by neutrophils, with IC(50) (microM) values of 2b, 2h, 2l, and 2o of 4.28+/-0.89, 3.31+/-0.88, 4.38+/-0.25, and 5.45+/-1.78, respectively. These results suggest that, in addition to the specific chromosomal aberrations and cell death, elevated apoptosis could also be a marker for exposure to anthracene derivatives.