Fabian Schumacher

1-Methoxy-3-indolylmethyl glucosinolate; a potent genotoxicant in bacterial and mammalian cells: Mechanisms of bioactivation

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, contained in many Brassica vegetables, is strongly mutagenic in Salmonella typhimurium TA100 when activated by myrosinase. Here, we describe the synthesis and evaluation of two breakdown products, 1-MIM nitrile and 1-MIM alcohol. 1-MIM nitrile was not mutagenic and 1-MIM alcohol showed low direct mutagenicity in TA100, indicating that other breakdown products mediated the mutagenicity of 1-MIM glucosinoate/myrosinase in this strain. However, 1-MIM alcohol was strongly mutagenic to a TA100-derived strain expressing human sulphotransferase SULT1A1. Likewise, 1-MIM glucosinolate (with myrosinase) showed 10 times higher mutagenic activity in TA100-SULT1A1 than in strain TA100. Identical adducts, N(2)-(1-MIM)-dG and N(6)-(1-MIM)-dA, were detected in both strains, but the levels were higher in TA100-hSULT1A1. A similar influence of SULT1A1 was observed in recombinant V79-hSULT1A1 cells compared to parental SULT-deficient Chinese hamster V79 cells. 1-MIM glucosinolate (with myrosinase) as well as 1-MIM alcohol induced sister chromatid exchange in both cell lines, but with clearly higher efficiency in V79-hSULT1A1 cells. Gene mutation assays were conducted at the HPRT locus with 1-MIM alcohol in V79-hSULT1A1 cells, and with 1-MIM glucosinolate/myrosinase in V79 parental cells. In both cases, the result was clearly positive. Thus, 1-MIM glucosinolate is mutagenic in bacterial and mammalian cells via at least two different metabolites.

Simultaneous Detection of Multiple DNA Adducts in Human Lung Samples by Isotope-Dilution UPLC-MS/MS

Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using liquid chromatography connected to tandem mass spectrometry (LC-MS/MS). However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from 16 ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by ultra performance liquid chromatography (UPLC)-MS/MS. Methods for the enrichment of the analytes from DNA hydrolysates and chromatographic separation preceding mass spectrometric analysis were optimized, and the resultant technique was used for the simultaneous analysis of the 16 DNA adducts in human lung biopsy specimens. Eleven adducts (formed by benzo[a]pyrene, 1-methylpyrene, 4-aminobiphenyl, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 1-methoxy-3-indolylmethylglucosinolate, 5-hydroxymethylfurfural, and malondialdehyde) were not detected in any tissue sample (limits of detection: 0.02–7.1 adducts/108 nucleosides). 3,N4-etheno-2′-deoxycytidine and 1,N6-etheno-2′-deoxyadenosine, formed from 2,3-epoxyaldehydes of endogenous lipid peroxidation products, were present in all subjects (16.9–115.3 and 27.2–179/108 nucleosides, respectively). The same was true for N2-(trans-methylisoeugenol-3′-yl)-2′-deoxyguanosine, the major adduct of methyleugenol (1.7–23.7/108 nucleosides). A minor adduct of methyleugenol and two adducts of furfuryl alcohol were detected in several pulmonary specimens. Taken together, we developed a targeted approach for the simultaneous mass spectrometric analyses of 16 DNA adducts, which can be easily extended by adducts formed from other mutagens. The method allowed one to detect adducts of furfuryl alcohol and methyleugenol in samples of human lung.

Ethyl cellulose nanocarriers and nanocrystals differentially deliver dexamethasone into intact, tape-stripped or sodium lauryl sulfate-exposed ex vivo human skin - assessment by intradermal microdialysis and extraction from the different skin layers.

Understanding penetration not only in intact, but also in lesional skin with impaired skin barrier function is important, in order to explore the surplus value of nanoparticle-based drug delivery for anti-inflammatory dermatotherapy. Herein, short-term ex vivo cultures of (i) intact human skin, (ii) skin pretreated with tape-strippings and (iii) skin pre-exposed to sodium lauryl sulfate (SLS) were used to assess the penetration of dexamethasone (Dex). Intradermal microdialysis was utilized for up to 24h after drug application as commercial cream, nanocrystals or ethyl cellulose nanocarriers applied at the therapeutic concentration of 0.05%, respectively. In addition, Dex was assessed in culture media and extracts from stratum corneum, epidermis and dermis after 24h, and the results were compared to those in heat-separated split skin from studies in Franz diffusion cells. Providing fast drug release, nanocrystals significantly accelerated the penetration of Dex. In contrast to the application of cream and ethyl cellulose nanocarriers, Dex was already detectable in eluates after 6h when applying nanocrystals on intact skin. Disruption of the skin barrier further accelerated and enhanced the penetration. Encapsulation in ethyl cellulose nanocarriers delayed Dex penetration. Interestingly, for all formulations highly increased concentrations in the dialysate were observed in tape-stripped skin, whereas the extent of enhancement was less in SLS-exposed skin. The results were confirmed in tissue extracts and were in line with the predictions made by in vitro release studies and ex vivo Franz diffusion cell experiments. The use of 45kDa probes further enabled the collection of inflammatory cytokines. However, the estimation of glucocorticoid efficacy by Interleukin (IL)-6 and IL-8 analysis was limited due to the trauma induced by the probe insertion. Ex vivo intradermal microdialysis combined with culture media analysis provides an effective, skin-sparing method for preclinical assessment of novel drug delivery systems at therapeutic doses in models of diseased skin.

Formulation and ex vivo evaluation of polymeric nanoparticles for controlled delivery of corticosteroids to the skin and the corneal epithelium

&NA; Controlled delivery of corticosteroids using nanoparticles to the skin and corneal epithelium may reduce their side effects and maximize treatment effectiveness. Dexamethasone‐loaded ethyl cellulose, Eudragit® RS and ethyl cellulose/Eudragit® RS nanoparticles were prepared by the solvent evaporation method. Dexamethasone release from the polymeric nanoparticles was investigated in vitro using Franz diffusion cells. Drug penetration was also assessed ex vivo using excised human skin. Nanoparticle toxicity was determined by MTT and H2DCFDA assays. Eudragit® RS nanoparticles were smaller and positively charged but had a lower dexamethasone loading capacity (0.3–0.7%) than ethyl cellulose nanoparticles (1.4–2.2%). By blending the two polymers (1:1), small (105 nm), positively charged (+37 mV) nanoparticles with sufficient dexamethasone loading (1.3%) were obtained. Dexamethasone release and penetration significantly decreased with decreasing drug to polymer ratio and increased when Eudragit® RS was blended with ethyl cellulose. Ex vivo, drug release and penetration from the nanoparticles was slower than a conventional cream. The nanoparticles bear no toxicity potentials except ethyl cellulose nanoparticles had ROS generation potential at high concentration. In conclusion, the nanoparticles showed great potential to control the release and penetration of corticosteroids on the skin and mucus membrane and maximize treatment effectiveness. Graphical abstract Figure. No caption available.

Detection of DNA Adducts Originating from 1-Methoxy-3-indolylmethyl Glucosinolate Using Isotope-Dilution UPLC-ESI-MS/MS

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, present at substantial levels in several food crops (e.g., broccoli and cabbage), forms DNA adducts in vitro and is mutagenic to bacterial and mammalian cells after activation by the plant enzyme myrosinase. Moreover, a breakdown product, 1-MIM alcohol, is metabolized to a secondary reactive intermediate by some mammalian sulfotransferases (SULTs). First, we incubated herring-sperm DNA with 1-MIM glucosinolate in the presence of myrosinase. We identified and synthesized the predominant adducts, N(2)-(1-MIM)-dG and N(6)-(1-MIM)-dA, and developed an UPLC-ESI-MS/MS method for their specific detection using isotopic dilution. Second, we demonstrated both DNA adducts in target cells (Salmonella typhimurium TA100 and Chinese hamster V79) of standard mutagenicity tests treated with 1-MIM glucosinolate/myrosinase as well as in 1-MIM alcohol-treated Salmonella and V79 cells engineered for expression of human SULT1A1. Similar excesses of N(2)-(1-MIM)-dG over N(6)-(1-MIM)-dA adducts were found in all cellular models independent of the test compound (1-MIM glucosinolate or alcohol), whereas dA adducts predominated in the cell-free system. Finally, we detected both DNA adducts in colon tissue of a mouse orally treated with 1-MIM glucosinolate. We are going to use this specific and sensitive method for investigating genotoxic risks of food-borne exposure to 1-MIM glucosinolate in animal and human studies.

In silico prediction of human sulfotransferase 1E1 activity guided by pharmacophores from molecular dynamics simulations

Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model, and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28% of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.

Biocompatibility and characterization of polyglycerol-based thermoresponsive nanogels designed as novel drug-delivery systems and their intracellular localization in keratinocytes

Abstract Novel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the stratum corneum and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG_dPG_tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG_dPG_pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery.

Enhanced Acid Sphingomyelinase Activity Drives Immune Evasion and Tumor Growth in Non–Small Cell Lung Carcinoma

The lipid hydrolase enzyme acid sphingomyelinase (ASM) is required for the conversion of the lipid cell membrane component sphingomyelin into ceramide. In cancer cells, ASM-mediated ceramide production is important for apoptosis, cell proliferation, and immune modulation, highlighting ASM as a potential multimodal therapeutic target. In this study, we demonstrate elevated ASM activity in the lung tumor environment and blood serum of patients with non-small cell lung cancer (NSCLC). RNAi-mediated attenuation of SMPD1 in human NSCLC cells rendered them resistant to serum starvation-induced apoptosis. In a murine model of lung adenocarcinoma, ASM deficiency reduced tumor development in a manner associated with significant enhancement of Th1-mediated and cytotoxic T-cell-mediated antitumor immunity. Our findings indicate that targeting ASM in NSCLC can act by tumor cell-intrinsic and -extrinsic mechanisms to suppress tumor cell growth, most notably by enabling an effective antitumor immune response by the host. Cancer Res; 77(21); 5963-76. ©2017 AACR.

Glucosinolates Are Mainly Absorbed Intact in Germfree and Human Microbiota-Associated Mice

Chemoprotective or genotoxic effects of glucosinolates occurring in Brassica vegetables are attributed to their hydrolysis products formed upon tissue damage by plant myrosinase. Since Brassica vegetables, in which myrosinase has been heat-inactivated, still display bioactivity, glucosinolate activation has been attributed to intestinal bacteria. The aim of this study was to investigate whether this is true. Glucoraphanin (172 mg/kg body weight) and neoglucobrassicin (297 mg/kg body weight) were administered intragastrically to germ free and human microbiota associated (HMA) mice. Approximately 30% of the applied doses of glucoraphanin and neoglucobrassicin were excreted unchanged in the urine of both germ free and HMA mice. Isothiocyanates, sulforaphane, and erucin, formed from glucoraphanin, were mainly excreted as urinary N-acetyl-l-cysteine conjugates. N-Methoxyindole-3-carbinol formed from neoglucobrassicin was observed in small amounts in both germ free and HMA mice. Formation of DNA adducts from neoglucobrassicin was also independent from bacterial colonization of the mice. Hence, intestinal bacteria are involved in the bioactivation of glucosinolates in the gut, but their contribution to glucosinolate transformation in HMA mice is apparently very small.

Method to Simultaneously Determine the Sphingosine 1-Phosphate Breakdown Product (2E)-Hexadecenal and Its Fatty Acid Derivatives Using Isotope-Dilution HPLC–Electrospray Ionization–Quadrupole/Time-of-Flight Mass Spectrometry

Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes, can be irreversibly degraded by the membrane-bound S1P lyase (S1PL) yielding (2E)-hexadecenal and phosphoethanolamine. It is discussed that (2E)-hexadecenal is further oxidized to (2E)-hexadecenoic acid by the long-chain fatty aldehyde dehydrogenase ALDH3A2 (also known as FALDH) prior to activation via coupling to coenzyme A (CoA). Inhibition or defects in these enzymes, S1PL or FALDH, result in severe immunological disorders or the Sjögren-Larsson syndrome, respectively. Hence, it is of enormous importance to simultaneously determine the S1P breakdown product (2E)-hexadecenal and its fatty acid metabolites in biological samples. However, no method is available so far. Here, we present a sensitive and selective isotope-dilution high performance liquid chromatography-electrospray ionization-quadrupole/time-of-flight mass spectrometry method for simultaneous quantification of (2E)-hexadecenal and its fatty acid metabolites following derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. Optimized conditions for sample derivatization, chromatographic separation, and MS/MS detection are presented as well as an extensive method validation. Finally, our method was successfully applied to biological samples. We found that (2E)-hexadecenal is almost quantitatively oxidized to (2E)-hexadecenoic acid, that is further activated as verified by cotreatment of HepG2 cell lysates with (2E)-hexadecenal and the acyl-CoA synthetase inhibitor triacsin C. Moreover, incubations of cell lysates with deuterated (2E)-hexadecenal revealed that no hexadecanoic acid is formed from the aldehyde. Thus, our method provides new insights into the sphingolipid metabolism and will be useful to investigate diseases known for abnormalities in long-chain fatty acid metabolism, e.g., the Sjögren-Larsson syndrome, in more detail.