Macranthoside B,a hederagenin saponin extracted from Lonicera macranthoides and its anti-tumor activities in vitro and in vivo

Macranthoside B (MB) is a hederagenin saponin extracted from the flower bud of Lonicera macranthoides. In this study, we defined the anticancer effect of MB both in vitro and in vivo using cell proliferation assays and xenograft tumor growth assays. Our data indicate that MB inhibits the proliferation of various kinds of cancer cells with IC₅₀ values in the range of 10–20 μM. Moreover, the volume and weight of xenograft tumors in nude mice treated with 5 mg/kg MB were decreased remarkably compared to those of the vehicle control group. Furthermore, DAPI staining and flow cytometry analysis with Annexin V/PI double staining revealed that more apoptotic cells were observed following MB treatment. In addition, degradation of PARP (poly-ADP-ribose polymerase), and activation of the caspase cascade for intrinsic pathways were observed. We also found that the expression of Bcl-2 protein decreased and the protein level of Bax increased which leading to an increase of the Bax/Bcl-2 ratio. Our results showed that MB exhibited strong anti-tumor effect and mitochondrion-mediated apoptosis induction involved in it.     

Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro

Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. We have investigated the role of AST in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis of the substantia nigra (SN) neurons in the mouse model of Parkinson’s disease (PD) and 1-methyl-4-phenylpyridinium (MPP+)-induced cytotoxicity of SH-SY5Y human neuroblastoma cells. In in vitro study, AST inhibits MPP+-induced production of intracellular reactive oxygen species (ROS) and cytotoxicity in SH-SY5Y human neuroblastoma cells. Preincubation of AST (50 μM) significantly attenuates MPP+-induced oxidative damage. Furthermore, AST is able to enhance the expression of Bcl-2 protein but reduce the expression of α-synuclein and Bax, and suppress the cleavage of caspase-3. Our results suggest that the protective effects of AST on MPP+-induced apoptosis may be due to its anti-oxidative properties and anti-apoptotic activity via induction of expression of superoxide dismutase (SOD) and catalase and regulating the expression of Bcl-2 and Bax. Pretreatment with AST (30 mg/kg) markedly increases tyrosine hydroxylase (TH)-positive neurons and decreases the argyrophilic neurons compared with the MPTP model group. In summary, AST shows protection from MPP+/MPTP-induced apoptosis in the SH-SY5Y cells and PD model mouse SN neurons, and this effect may be attributable to upregulation of the expression of Bcl-2 protein, downregulation of the expression of Bax and α-synuclein, and inhibition of the activation of caspase-3. These data indicate that AST may provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson’s disease.     

Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure

Methylmercury (MeHg) is an environmental neurotoxicant which induces neuropathological changes in both the central nervous and peripheral sensory nervous systems. Our recent study demonstrated that down-regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1), which is known to promote neuritic extension, preceded MeHg-induced damage in cultured cortical neurons, suggesting that MeHg-mediated axonal degeneration is due to the disturbance of neuritic extension. Therefore we hypothesized that MeHg-induced axonal degeneration might be caused by neuritic extension/retraction incoordination. This idea brought our attention to the Ras homolog gene (Rho)/Rho-associated coiled coil-forming protein kinase (ROCK) pathway because it has been known to be associated with the development of axon and apoptotic neuronal cell death. Here we show that inhibition of the Rho/ROCK pathway prevents MeHg-intoxication both in vitro and in vivo. A Rho inhibitor, C3 toxin, and 2 ROCK inhibitors, Fasudil and Y-27632, significantly protected against MeHg-induced axonal degeneration and apoptotic neuronal cell death in cultured cortical neuronal cells exposed to 100 nM MeHg for 3 days. Furthermore, Fasudil partially prevented the loss of large pale neurons in dorsal root ganglia, axonal degeneration in dorsal spinal root nerves, and vacuolar degeneration in the dorsal columns of the spinal cord in MeHg-intoxicated model rats (20 ppm MeHg in drinking water for 28 days). Hind limb crossing sign, a characteristic MeHg-intoxicated sign, was significantly suppressed in this model. The results suggest that inhibition of the Rho/ROCK pathway rescues MeHg-mediated neuritic extension/retraction incoordination and is effective for the prevention of MeHg-induced axonal degeneration and apoptotic neuronal cell death.     

Antitumor effects of emodin on LS1034 human colon cancer cells in vitro and in vivo: Roles of apoptotic cell death and LS1034 tumor xenografts model

Emodin, an active natural anthraquinone derivative, is found in the roots and rhizomes of numerous Chinese medicinal herbs and exhibits anticancer effects on many types of human cancer cell lines. The aim of this study investigated that emodin induced apoptosis of human colon cancer cells (LS1034) in vitro and inhibited tumor nude mice xenografts bearing LS1034 in vivo. In in vitro study, emodin induced cell morphological changes, decreased the percentage of viability, induced G2/M phase arrest and increased ROS and Ca²⁺ productions as well as loss of mitochondrial membrane potential (ΔΨ_m) in LS1034 cells. Emodin-triggered apoptosis was also confirmed by DAPI staining and these effects are concentration-dependent. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9 and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. Emodin induced the productions of ROS and Ca²⁺ release, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in tumor nude mice xenografts bearing LS1034. Overall, the potent in vitro and in vivo antitumor activities of emodin suggest that it might be developed for treatment of colon cancer in the future.     

Gellan gum blended PEI nanocomposites as gene delivery agents: Evidences from in vitro and in vivo studies

Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siRNA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.     

Formulation and in vitro/in vivo evaluation of levodopa transdermal delivery systems

This study aims to investigate the feasibility of Levodopa transdermal delivery systems (TDSs). Levodopa TDSs were formulated using various vehicles and permeation enhancers, and in vitro permeation and in vivo pharmacokinetic studies were carried out. In the in vitro study, ester-type vehicles showed relatively high enhancing effects; propylene glycol monocaprylate and propylene glycol monolaurate showed the highest permeation fluxes from both solution and pressure sensitive adhesive (PSA) TDS formulations. Lag time was dramatically shortened with PSA TDS formulations as compared with solution formulations. In the in vivo study, the addition of fatty acids increased blood drug concentrations regardless of the kind or concentration of fatty acid; the AUC_inf increased up to 8.7 times as compared with propylene glycol (PG) alone. PSA TDS containing 10% linoleic acid exhibited prolonged T_max as compared with oral form. Total clearance of L-dopa from PSA TDSs was significantly lower than from oral form (up to 86.8 times). Especially, PSA TDS containing 10% linoleic acid (LOA) revealed 76.2 fold higher AUC_inf than oral administration. Based on our results, the L-dopa PSA TDS containing PG with 10% LOA could be used as a good adjuvant therapy for Parkinson's disease patients who experience symptom fluctuation by L-dopa oral administration.     

LL-202, a newly synthesized flavonoid,inhibits tumor growth via inducing G2/M phase arrest and cell apoptosis in MCF-7 human breast cancer cells in vitro and in vivo

We recently established that LL-202, a newly synthesized flavonoid, exhibited obvious anticancer effects against human breast cells in vivo and in vitro. The underlying mechanism of its anticancer activity remains to be elucidated. In this study, we demonstrated that LL-202 inhibited the growth and proliferation of human breast cancer MCF-7 cells in a concentration and time-dependent manner. We reported that LL-202 induced both mitochondrial- and death-receptor-mediated apoptosis, which were characterized by the dissipation of mitochondrial membrane potential (ΔΨ_m), cytochrome c (Cyt c) release from mitochondria to cytosol, the activation of several caspases and induction of poly (ADP-ribose) polymerase (PARP) and Bid cleavage. N-acetylcysteine (NAC), a general ROS scavenger, partly blocked the LL-202-induced ROS levels and apoptosis. In addition, LL-202 induced arrest in cell cycle progression at G2/M phase in MCF-7 cells. After the treatment with LL-202, the expression of cell cycle-related proteins, such as cyclin B1, cyclin A, and p-CDK1 (Thr161) were down-regulated, whereas the expression of p21^WAF1/Cip1 and p-CDK1 (Thr14/Tyr15) were up-regulated. Finally, in vivo studies, LL-202 significantly suppressed the growth of MCF-7 breast cancer xenograft tumors in a dose-dependent manner with low systemic toxicity. In conclusion, the results showed that LL-202 had significant anticancer effects against human breast cells via the induction of apoptosis and G2/M phase arrest and it may be a novel anticancer agent for treatment of breast cancer.     

Exploring the in vitro formation of trimethylarsine sulfide from dimethylthioarsinic acid in anaerobic microflora of mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS

Although metabolism of arsenicals to form methylated oxoarsenical species has been extensively studied, less is known about the formation of thiolated arsenical species that have recently been detected as urinary metabolites. Indeed, their presence suggests that the metabolism of ingested arsenic is more complex than previously thought. Recent reports have shown that thiolated arsenicals can be produced by the anaerobic microflora of the mouse cecum, suggesting that metabolism prior to systemic absorption may be a significant determinant of the pattern and extent of exposure to various arsenic-containing species. Here, we examined the metabolism of ³⁴S labeled dimethylthioarsinic acid (³⁴S-DMTA^V) by the anaerobic microflora of the mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS/MS to monitor for the presence of various oxo- and thioarsenicals. The use of isotopically enriched ³⁴S-DMTA^V made it possible to differentiate among potential metabolic pathways for production of the trimethylarsine sulfide (TMAS^V). Upon in vitro incubation in an assay containing anaerobic microflora of mouse cecum, ³⁴S-DMTA^V underwent several transformations. Labile ³⁴S was exchanged with more abundant ³²S to produce ³²S-DMTA^V, a thiol group was added to yield DMDTA^V, and a methyl group was added to yield ³⁴S-TMAS^V. Because incubation of ³⁴S-DMTA^V resulted in the formation of ³⁴S-TMAS^V, the pathway for its formation must preserve the arsenic-sulfur bond. The alternative metabolic pathway postulated for formation of TMAS^V from dimethylarsinic acid (DMA^V) would proceed via a dimethylarsinous acid (DMA^III) intermediate and would necessitate the loss of ³⁴S label. Structural confirmation of the metabolic product was achieved using HPLC-ESI-MS/MS. The data presented support the direct methylation of DMTA^V to TMAS^V. Additionally, the detection of isotopically pure ³⁴S-TMAS^V raises questions about the sulfur exchange properties of TMAS^V in the cecum material. Therefore, ³⁴S-TMAS^V was incubated and the exchange was monitored with respect to time. The data suggest that the As-S bond associated with TMAS^V is less labile than the As-S bond associated with DMTA^V.     

The in vitro effects of Trolox on methylmercury-induced neurotoxicity

Methylmercury (MeHg), an environmental toxicant primarily found in fish and seafood poses a dilemma to both consumers and regulatory authorities given the nutritional benefits of fish consumption vs. possible adverse neurological damage caused by MeHg. The present study addresses whether supplementation with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), alters the neuro-oxidative effects of MeHg in C6-glioma and B35-neuronal cell lines. As indicators of cytotoxicity, reduced glutathione (GSH), reactive oxygen species (ROS) and mitochondrial activity (MTT) were measured. The cellular mercury (Hg) content was measured with high resolution-inductively coupled plasma mass spectrometry (HR-ICPMS). The amount of MeHg-induced ROS was significantly reduced (p < 0.05) after treatment with 50 μM Trolox in C6 glial cell line. However, treatment with Trolox did not induce any significant increase in GSH levels or MTT activity in either of the cell lines. In addition, treatment with Trolox did not induce any significant changes in intracellular MeHg levels. The MeHg and Trolox treated C6 glial cell line differed significantly (p < 0.05) from the B35 cell line for MTT, ROS and GSH activity. These findings provide experimental evidence that preincubation with Trolox prevents MeHg-induced ROS generation in C6 glial cell line by quenching of free radicals and not by changes in intracellular GSH or MeHg content.     

Generation,characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs

PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs — HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG₂₀₀₀ conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH₂CH₂) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics.     

Activation of P53 in HepG2 cells as surrogate to detect mutagens and promutagens in vitro

The current genotoxicity tests of the standard in vitro battery, especially those using mammalian cells, are limited by their low specificity and highlight the importance of new in vitro tools. This study aimed to evaluate the suitability of HepG2 cells for assaying mutagens and promutagens. We determined P53 activity as surrogate genotoxicity endpoint in HepG2 cells. Our results revealed a significant P53-induction by actinomycin D, methyl methanesulfonate and etoposide. Prior to the investigation of promutagens we characterized HepG2 cells by analyzing the expression of 45 genes involved in xenobiotic metabolism and measuring the activity of selected Cytochrome-P450 (CYP) enzymes. We determined a limited metabolic capacity prompting us to employ a co-treatment with rat liver S9 as metabolic activation system (MAS) for promutagens. While cyclophosphamide showed an elevation of activated P53 in the presence of S9, 7,12-dimethylbenz[a]anthracene and aflatoxin B₁ responded without the MAS. Inhibition of cellular CYP3A4 or CYP1A/1B suppressed the aflatoxin B₁- and dimethylbenz[a]anthracene-mediated P53 response, respectively, indicating that HepG2 cells are capable of metabolizing these compounds in a CYP1A/B/3A4-dependent manner. In summary, our results indicate that P53 activation in HepG2 cells combined with a MAS can be used for the identification of new (pro)genotoxicants.     

Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatin derivatives against human cancer cell lines and primary human hepatocytes

A series of seven platinum(II) cyclobutane-1,1-dicarboxylato (cbdc) complexes {[Pt(cbdc)(L_n)₂], 1-7}, derived from carboplatin by a substitution of two NH₃ molecules for two 2,6,9-trisubstituted 6-benzylaminopurine-based N-donor ligands (L_n), was studied by the MTT assay for their in vitro cytotoxic activity against seven human cancer cell lines, i.e. lung carcinoma (A549), cervix epithelioid carcinoma (HeLa), osteosarcoma (HOS), malignant melanoma (G361), breast adenocarcinoma (MCF7), ovarian carcinoma (A2780) and its cisplatin-resistant analogue (A2780cis), and against two primary cultures of human hepatocytes (LH31 and LH32). The prepared complexes were cytotoxic against several cancer cells, in some cases even more than cisplatin. The best results were achieved for complexes 1 (IC₅₀ = 17.4 ± 2.0 μM) and 2 (IC₅₀ = 14.8 ± 2.1 μΜ) against HOS cells, 1 (IC₅₀ = 15.1 ± 6.8 μM), 2 (IC₅₀ = 13.6 ± 5.2 μM) and 6 (IC₅₀ = 19.0 ± 6.6 μM) against MCF7, 6 (IC₅₀ = 6.4 ± 0.1 μM) against A2780, and 1-6 (IC₅₀ = 15.6 ± 4.0, 12.9 ± 3.7, 15.8 ± 3.8, 16.6 ± 5.5, 22.1 ± 2.5, and 5.6 ± 1.7 μM, respectively) against A2780cis. Viability of human hepatocytes was not declined by the tested complexes up to the concentration of 50 μM (for 1, 3-7) and 20 μM (for 2; caused by lower solubility of this complex).     

Elimination pathways of cyclosarin (GF) mediated by β-cyclodextrin in vitro: Pharmacokinetic and toxicokinetic aspects

Cyclodextrins (CD) are promising small molecular scavengers showing favourable degradation of extremely toxic organophosphorus compounds (OP) such as tabun (GA), soman (GD) or cyclosarin (GF). For β-CD derivatives as potential OP antidotes with low intrinsic toxicity it is of great interest to completely understand the modes of interaction of both compounds in terms of OP detoxification. The mechanisms of CD action are not completely understood which prompted us to investigate the interactions of GF and β-cyclodextrin (β-CD) as model compounds. Using positive electrospray ionization mass spectrometry (ESI/MS), the formation of covalent conjugates of β-CD with O-cyclohexylmethylphosphonate (CHMP) residue was detected for the first time and was examined in vitro. With a newly developed LC–MS method the formation of O-cyclohexylmethylphosphonic acid (CHMPA) (i.e. GF hydrolysis) and covalent CHMP-β-CD conjugates was analyzed. Compared to water, tris(hydroxymethyl)aminomethane (TRIS) reduced the formation of covalent conjugates but amplified formation of CHMPA. Depending on experimental conditions the degradation of GF by β-CD may be preferably catalytic or stoichiometric. For illustrating different possible reaction pathways a scheme was established that could support the idea of β-CD acting as an artificial enzyme. These results provide an important insight into the β-CD mediated detoxification pathways of GF.     

In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450

The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC₅₀) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC₅₀ = 412.68 ± 15.44 μM) and CYP3A4 (IC₅₀ = 343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC₅₀ = 539.04 ± 14.18 μM) and CYP3A4 (IC₅₀ = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (K_i = 385.24 ± 8.75 μM) and CYP3A4 (K_i = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (K_i = 109.62 ± 6.14 μM) and CYP3A4 (K_i = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.     

Identification of nevadensin as an important herb-based constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect

Estragole is a natural constituent of several herbs and spices including sweet basil. In rodent bioassays, estragole induces hepatomas, an effect ascribed to estragole bioactivation to 1′-sulfooxyestragole resulting in DNA adduct formation. The present paper identifies nevadensin as a basil constituent able to inhibit DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1′-hydroxyestragole and nevadensin. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1′-hydroxyestragole. The Ki for SULT inhibition by nevadensin was 4 nM in male rat and human liver fractions. Furthermore, nevadensin up to 20 μM did not inhibit 1′-hydroxyestragole detoxification by glucuronidation and oxidation. The inhibition of SULT by nevadensin was incorporated into the recently developed physiologically based biokinetic (PBBK) rat and human models for estragole bioactivation and detoxification. The results predict that co-administration of estragole at a level inducing hepatic tumors in vivo (50 mg/kg bw) with nevadensin at a molar ratio of 0.06, representing the ratio of their occurrence in basil, results in almost 100% inhibition of the ultimate carcinogen 1′-sulfooxyestragole when assuming 100% uptake of nevadensin. Assuming 1% uptake, inhibition would still amount to more than 83%. Altogether these data point at a nevadensin-mediated inhibition of the formation of the ultimate carcinogenic metabolite of estragole, without reducing the capacity to detoxify 1′-hydroxyestragole via glucuronidation or oxidation. These data also point at a potential reduction of the cancer risk when estragole exposure occurs within a food matrix containing SULT inhibitors compared to what is observed upon exposure to pure estragole.     

Effects of T-2 toxin and selenium on chondrocyte expression of matrix metalloproteinases (MMP-1, MMP-13), α2-macroglobulin (α2M) and TIMPs

T-2 toxin is regarded as an important etiological factor of Kashin-Beck disease, and supplementation of selenium-salt partly prevents Kashin-Beck disease. The present study investigated the effects of T-2 toxin on the degradation of type II collagen in human chondrocytes in vitro. Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without T-2 toxin and selenium. Immunohistochemistry analyses showed that T-2 toxin decreased type II collagen staining and selenium appeared to prevent the decrease in type II collagen induced by T-2 toxin in engineered cartilage. Then, Western blot and RT-PCR analyses showed that an increase in MMP-13 and MMP-1 expressions, and a decrease in the expression of the general endoproteinase inhibitor (α₂M) were induced by T-2 toxin. Gelatin reverse zymography showed that TIMP-1 and TIMP-2 levels were decreased in a dose-dependent manner after exposure of T-2 toxin. Selenium had a protective role by increasing the level of type II collagen protein through down-regulation of MMP-13 protein and mRNA expression and up-regulation of TIMP-1 and TIMP-2 expressions. These data suggest T-2 toxin induces cartilage matrix degradation by the up-regulation of MMP-13 and TIMP-1, and down-regulation of TIMP-2 and α₂M expressions.     

Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity

Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial LPS co-administration to induce an inflammatory background have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs. However, the low-throughput nature of these models renders them problematic for employment as preclinical screening assays. Here, we present an analogous, but high-throughput, in vitro approach in which drugs are administered to a variety of cell types (primary human and rat hepatocytes and the human HepG2 cell line) across a landscape of inflammatory contexts containing LPS and cytokines TNF, IFNγ, IL-1α, and IL-6. Using this assay, we observed drug-cytokine hepatotoxicity synergies for multiple idiosyncratic hepatotoxicants (ranitidine, trovafloxacin, nefazodone, nimesulide, clarithromycin, and telithromycin) but not for their corresponding non-toxic control compounds (famotidine, levofloxacin, buspirone, and aspirin). A larger compendium of drug-cytokine mix hepatotoxicity data demonstrated that hepatotoxicity synergies were largely potentiated by TNF, IL-1α, and LPS within the context of multi-cytokine mixes. Then, we screened 90 drugs for cytokine synergy in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19%) synergized with a single cytokine mix than did the non-hepatotoxic drugs (3%). Finally, we used an information theoretic approach to ascertain especially informative subsets of cytokine treatments for most highly effective construction of regression models for drug- and cytokine mix-induced hepatotoxicities across these cell systems. Our results suggest that this drug-cytokine co-treatment approach could provide a useful preclinical tool for investigating inflammation-associated idiosyncratic drug hepatotoxicity.