Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

Conformation-dependent lesion bypass of bulky arylamine-dG adducts generated from 2-nitrofluorene in epigenetic sequence contexts.

Sequence context influences structural characteristics and repair of DNA adducts, but there is limited information on how epigenetic modulation affects conformational heterogeneity and bypass of DNA lesions. Lesions derived from the environmental pollutant 2-nitrofluorene have been extensively studied as chemical carcinogenesis models; they adopt a sequence-dependent mix of two significant conformers: major groove binding (B) and base-displaced stacked (S). We report a conformation-dependent bypass of the N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (dG-FAF) lesion in epigenetic sequence contexts (d[5'-CTTCTC#G*NCCTCATTC-3'], where C# is C or 5-methylcytosine (5mC), G* is G or G-FAF, and N is A, T, C or G). FAF-modified sequences with a 3' flanking pyrimidine were better bypassed when the 5' base was 5mC, whereas sequences with a 3' purine exhibited the opposite effect. The conformational basis behind these variations differed; for -CG*C- and -CG*T-, bypass appeared to be inversely correlated with population of the duplex-destabilizing S conformer. On the other hand, the connection between conformation and a decrease in bypass for flanking purines in the 5mC sequences relative to C was more complex. It could be related to the emergence of a disruptive non-S/B conformation. The present work provides novel conformational insight into how 5mC influences the bypass efficiency of bulky DNA damage.

Copper Sulfide Facilitates Hepatobiliary Clearance of Gold Nanoparticles through the Copper-Transporting ATPase ATP7B.

Metallic gold (Au) nanoparticles have great potential for a wide variety of biomedical applications. Yet, slow clearance of Au nanoparticles significantly hinders their clinical translation. Herein, we describe a strategy of utilizing the endogenous copper (Cu) clearance to improve the elimination of Au nanoparticles. Our mechanistic study reveals that a Cu-transporting P-type ATPase, ATP7B, mediates the exocytosis of CuS nanoparticles into bile canaliculi for their rapid hepatobiliary excretion. The efflux of CuS nanoparticles is adopted to facilitate the hepatobiliary clearance of Au nanoparticles through CuS-Au conjugation. Using two different CuS-Au nanoconjugates, we demonstrate that CuS increases the biliary Au excretion of CuS-Au nanospheres or CuS-Au nanorods in mice or rats in comparison to that of their respective unconjugated Au nanoparticles postintravenous injection. The current CuS-Au conjugation approach provides a feasible strategy to enhance the hepatobiliary clearance of Au nanoparticles that may be applicable to various structures.

Enhanced reactive oxygen species through direct copper sulfide nanoparticle-doxorubicin complexation.

CuS-based nanostructures loading the chemotherapeutic agent doxorubicin (DOX) exerted excellent cancer photothermal chemotherapy under multi-external stimuli. The DOX loading was generally designed through electrostatic interaction or chemical linkers. However, the interaction between DOX molecules and CuS nanoparticles has not been investigated. In this work, we use PEGylated hollow copper sulfide nanoparticles (HCuSNPs) to directly load DOX through the DOX/Cu2+ chelation process. Distinctively, the synthesized PEG-HCuSNPs-DOX release the DOX/Cu2+ complexes into surrounding environment, which generate significant reactive oxygen species (ROS) in a controlled manner by near-infrared laser. The CuS nanoparticle-mediated photothermal ablation facilitates the ROS-induced cancer cell killing effect. Our current work reveals a DOX/Cu2+-mediated ROS-enhanced cell-killing effect in addition to conventional photothermal chemotherapy through the direct CuS nanoparticle-DOX complexation.

Direct Dry-Grinding Synthesis of Monodisperse Lipophilic CuS Nanoparticles.

Copper sulfide nanoparticles, effective absorbers of near-infrared light, are recently attracting broad interest as a photothermal coupling agent for cancer therapy. Lipophilic copper sulfide nanoparticles are preferred for high performance biomedical applications due to high tissue affinity. Synthesis of lipophilic copper sulfide nanoparticles requires complicated multi-step processes under severe conditions. Here, we describe a new synthetic process, developed by direct dry-grinding of copper(II) acetylacetonate with sulfur under ambient environment at low temperature. The formed CuS nanoparticles are of uniform size, ~10 nm in diameter, and are monodispersed in chloroform. Each covellite CuS nanocrystal surface is modified with oleylamine through hydrogen bonding between sulfur atoms and amine groups of oleylamine. The nanoparticles demonstrate near-infrared light absorption for photothermal applications. The synthetic methodology described here is more convenient and less extreme than previous methods, and should thus greatly facilitate the preparation of photothermal lipophilic copper sulfide nanomaterials for cancer therapy.

Ontogenic expression of human carboxylesterase-2 and cytochrome P450 3A4 in liver and duodenum: postnatal surge and organ-dependent regulation.

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner.

A comparative study of hollow copper sulfide nanoparticles and hollow gold nanospheres on degradability and toxicity.

Gold and copper nanoparticles have been widely investigated for photothermal therapy of cancer. However, degradability and toxicity of these nanoparticles remain concerns. Here, we compare hollow CuS nanoparticles (HCuSNPs) with hollow gold nanospheres (HAuNS) in similar particle sizes and morphology following intravenous administration to mice. The injected pegylated HCuSNPs (PEG-HCuSNPs) are eliminated through both hepatobiliary (67 percentage of injected dose, %ID) and renal (23 %ID) excretion within one month postinjection. By contrast, 3.98 %ID of Au is excreted from liver and kidney within one month after iv injection of pegylated HAuNS (PEG-HAuNS). Comparatively, PEG-HAuNS are almost nonmetabolizable, while PEG-HCuSNPs are considered biodegradable nanoparticles. PEG-HCuSNPs do not show significant toxicity by histological or blood chemistry analysis. Principal component analysis and 2-D peak distribution plots of data from matrix-assisted laser desorption ionization-time-of-flight imaging mass spectrometry (MALDI-TOF IMS) of liver tissues demonstrated a reversible change in the proteomic profile in mice receiving PEG-HCuSNPs. This is attributed to slow dissociation of Cu ion from CuS nanoparticles along with effective Cu elimination for maintaining homeostasis. Nonetheless, an irreversible change in the proteomic profile is observed in the liver from mice receiving PEG-HAuNS by analysis of MALDI-TOF IMS data, probably due to the nonmetabolizability of Au. This finding correlates with the elevated serum lactate dehydrogenase at 3 months after PEG-HAuNS injection, indicating potential long-term toxicity. The comparative results between the two types of nanoparticles will advance the development of HCuSNPs as a new class of biodegradable inorganic nanomaterials for photothermal therapy.

Antioxidant sulforaphane and sensitizer trinitrobenzene sulfonate induce carboxylesterase-1 through a novel element transactivated by nuclear factor-E2 related factor-2.

Carboxylesterase-1 (CES1), the most versatile human carboxylesterase, plays critical roles in drug metabolism and lipid mobilization. This enzyme is highly induced by antioxidants and sensitizers in various cell lines. These compounds are known to activate nuclear factor-E2 related factor-2 (Nrf2) by reacting to kelch-like ECH-associated protein-1 (Keap1). The aims of this study were to determine whether antioxidant sulforaphane (SFN) and sensitizer trinitrobenzene sulfonate (TNBS) target Keap1 similarly and whether they use the same element for CES1 induction. Cells over-expressing Keap1 were treated with TNBS or SFN and the formation of disulfide bonds among Keap1 molecules were determined. SFN promoted intramolecular disulfide formation whereas TNBS promoted intermolecular disulfide formation of Keap1. Two elements, sensitizing/antioxidant response element (S/ARE) and ARE4, were identified to support Nrf2 in the regulated expression of CES1A1. Both elements were bound by Nrf2, however, the S/ARE element supported, whereas the ARE4 element repressed Nrf2 transactivation. The repression required higher amounts of Nrf2, suggesting that the transactivation through the S/ARE element dominates the trans-repression through the ARE4 element under normal antioxidative condition. These findings conclude that compounds, although triggering the Keap1-Nrf2 pathway, may differ in the mode of reacting with Keap1. These findings also conclude that both positive and negative Nrf2 elements exist even within the same gene, and such opposing mechanisms provide fine-tuning in transcriptional regulation by the Keap1-Nrf2 pathway. High levels of CES1 are linked to lipid retention. Excessive induction of CES1 by antioxidants and sensitizers likely provides a mechanism for potential detrimental effect on human health.

Age-related inducibility of carboxylesterases by the antiepileptic agent phenobarbital and implications in drug metabolism and lipid accumulation.

Carboxylesterases (CES) constitute a class of hydrolytic enzymes that play critical roles in drug metabolism and lipid mobilization. Previous studies with a large number of human liver samples have suggested that the inducibility of carboxylesterases is inversely related with age. To directly test this possibility, neonatal (10 days of age) and adult mice were treated with the antiepileptic agent phenobarbital. The expression and hydrolytic activity were determined on six major carboxylesterases including ces1d, the ortholog of human CES1. Without exception, all carboxylesterases tested were induced to a greater extent in neonatal than adult mice. The induction was detected at mRNA, protein and catalytic levels. Ces1d was greatly induced and found to rapidly hydrolyze the antiplatelet agent clopidogrel and support the accumulation of neutral lipids. Phenobarbital represents a large number of therapeutic agents that induce drug metabolizing enzymes and transporters in a species-conserved manner. The higher inducibility of carboxylesterases in the developmental age likely represents a general phenomenon cross species including human. Consequently, individuals in the developmental age may experience greater drug-drug interactions. The greater induction of ces1d also provides a molecular explanation to the clinical observation that children on antiepileptic drugs increase plasma lipids.

Hypolipidemic agent Z-guggulsterone: metabolism interplays with induction of carboxylesterase and bile salt export pump.

Z-Guggulsterone is a major ingredient in the Indian traditional hypolipidemic remedy guggul. A study in mice has established that its hypolipidemic effect involves the farnesoid X receptor (FXR), presumably by acting as an antagonist of this receptor. It is generally assumed that the antagonism leads to induction of cytochrome P450 7A1 (CYP7A1), the rate-limiting enzyme converting free cholesterol to bile acids. In this study, we tested whether Z-guggulsterone indeed induces human CYP7A1. In addition, the expression of cholesteryl ester hydrolase CES1 and bile salt export pump (BSEP) was monitored. Contrary to the general assumption, Z-guggulsterone did not induce CYP7A1. Instead, this phytosterol significantly induced CES1 and BSEP through transactivation. Z-Guggulsterone underwent metabolism by CYP3A4, and the metabolites greatly increased the induction potency on BSEP but not on CES1. BSEP induction favors cholesterol elimination, whereas CES1 involves both elimination and retention (probably when excessively induced). Interestingly, clinical trials reported the hypolipidemic response rates from 18% to 80% and showed that higher dosages actually increased VLDL cholesterol. Our findings predict that better hypolipidemic outcomes likely occur in individuals who have a relatively higher capacity of metabolizing Z-guggulsterone with moderate CES1 induction, a scenario possibly achieved by lowering the dosing regimens.

Pyrethroid insecticides: isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor.

Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.