Induction and quail liver diamine oxidase (histaminase). Part II: Different responses to the inducers

In quail liver microsomes the metabolism of histamine is prevalently sustained by diaminoxidase (DAO). Treatment with phenobarbital (PB) and clofibrate (CF) does not modify DAO activity, while that with Aroclor 1254 (PCB) and beta-naphtoflavone (BNF) significantly decreases it. The decrease of DAO activity produced by these substances is not dependent on their direct inhibition of the enzyme. DAO decrease might represent a further possibility to discriminate among different inducers; moreover, it implies that histamine metabolism might be decreased, in quail liver, by some classes of inducers.     

Clinical observation on YiSuiShengXueGranule on treating 156 patients with beta-thalassemia major and the molecular mechanism study

OBJECTIVE: To investigate the clinical effects and security of YiSuiShengXueGranule (YSSXG) on treating 156 patients with beta-thalassemia major. Methods: YSSXG was given orally to 156 patients with beta-thalassemia in GuangXi Autonomous Region (the high incidence area of beta-thalassemia in China) for 3 months as one therapeutic course, 3 times a day, 10 g each time (for children, the dose should be reduced properly according to their body weight and age), and no blood transfusion used during the course. Clinical symptoms and levels of hemoglobin (Hb), red blood cell (RBC), reticulocyte (Ret) and hemoglobin F (HbF) were observed before and after treatment, and side-effects were observed during the course. A 3-6 months follow up study was performed after withdrawal of YSSXG. And systemic gene analysis was conducted with PCR, SSCP-PCR, RT-PCR and DNA sequences analysis and mRNA differently expression technique, in order to study the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation. RESULTS: Levels of Hb, RBC, Ret and HbF obviously elevated, and clinical symptoms markedly ameliorated in patients after treated with YSSXG from the 1st to 3rd month (all p<0.01). Dynamical observation showed that the improvement of symptoms kept accordance with the elevation of hemorrheological indexes. The treatment was effective in 145 patients and ineffective in 11, and the total effective rate was 92.9%, without any adverse reaction founded. Follow-up studies showed the therapeutic effect could sustain for 3 to 4 months after drug-withdrawal. The molecular mechanism study showed: YSSXG did not change the genetic mutation type, but could obviously increase gamma/(beta+gamma) globin ratio, both gamma-globin mRNA and GM-CSF mRNA expression were significantly enhanced so as to induce HbF synthesis increasing after treated with YSSXG. CONCLUSION: YSSXG had obvious effects in treating beta-thalassemia by unlocking gamma-gene, increasing the gamma-globin expression and enhancing HbF synthesis so as to compensate for the gene defect. This study has provided a new path for the treatment of beta-thalassemia with Traditional Chinese Medicine.     

Alkylformamides as inducers of tumour cell differentiation--a mini-review

The induction of terminal differentiation in tumour cells represents a possible therapeutic strategy for treating cancer. The alkylformamides are 1 group of experimental compounds which have been shown to induce terminal differentiation in human HL-60 leukemia and murine Friend erythroleukemia cells in vitro. Their mechanism of action is unknown. Dimethylformamide has been used as a model inducer in carcinoma and fibroblastic models. Analysis of the relationship between structure and inducing activity of the alkylformamides in vitro reveals that no specificity of structure exists and that their properties as inducers of terminal differentiation extend to related compounds, e.g. the alkylacetamides and alkylureas. This is in contrast to the marked specificity of N-methylformamide (NMF) as an in vivo antitumour agent. The potency of these compounds as inducers of differentiation is predictable and correlated with their molecular weight. High concentrations of NMF are required to induce differentiation in vitro and these concentrations are not achievable in vivo. However, while NMF is unlikely to be a useful inducer in vivo many of its higher MW analogues are very much more potent as inducers in vitro and yet no more toxic (to the host) in vivo. Some of these (e.g. tetramethylurea or 1,3-dimethylurea) may be capable of achieving inducing concentrations in vivo.     

Assessment of hepatocytes and liver slices as in vitro test systems to predict in vivo gene expression.

The use of in vitro systems to predict in vivo responses to chemical agents provides the benefits of requiring fewer animals, reducing variability between samples, requiring less test material, and enabling higher throughput. In the present study rat tissue slices and primary hepatocytes were compared as in vitro systems to predict in vivo changes in gene expression in response to treatment with known liver toxicants or inducers. Five compounds (phenobarbital, carbon tetrachloride, Wy-14,634, alpha-napthylisothiocyanate, and tacrine) were chosen for their established and diverse mechanisms of hepatoxicity or microsomal induction. Expression profiles from male Sprague-Dawley rats or in vitro systems treated for 24 h were measured by DNA oligonucleotide microarrays containing 8700 probe sets. Qualitative comparison of expression revealed a >80% concordance between in vivo liver and both in vitro systems; however, the responsiveness of both in vitro systems to compound-induced changes in gene expression was far less than that of in vivo. Furthermore, both in vitro systems appeared similar in their ability to reproduce compound-induced changes in gene expression observed in vivo.     

Regulatory cross-talk between drug metabolism and lipid homeostasis: constitutive androstane receptor and pregnane X receptor increase Insig-1 expression

Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) by xenobiotic inducers of cytochromes P450 is part of a pleiotropic response that includes liver hypertrophy, tumor promotion, effects on lipid homeostasis, and energy metabolism. Here, we describe an acute response to CAR and PXR activators that is associated with induction of Insig-1, a protein with antilipogenic properties. We first observed that activation of CAR and PXR in mouse liver results in activation of Insig-1 along with reduced protein levels of the active form of sterol regulatory element binding protein 1 (Srebp-1). Studies in mice deficient in CAR and PXR revealed that the effect on triglycerides involves these two nuclear receptors. Finally, we identified a functional binding site for CAR and PXR in the Insig-1 gene by in vivo, in vitro, and in silico genomic analysis. Our experiments suggest that activation Insig-1 by drugs leads to reduced levels of active Srebp-1 and consequently to reduced target gene expression including the genes responsible for triglyceride synthesis. The reduction nuclear Srebp-1 by drugs is not observed when Insig-1 expression is repressed by small interfering RNA. In addition, observed that Insig-1 is also a target of AMP-activated kinase, the hepatic activity of which is increased by activators of CAR and PXR and is known to cause a reduction of triglycerides. The fact that drugs that serve as CAR or PXR ligands induce Insig-1 might have clinical consequences and explains alterations lipid levels after drug therapy.     

Effects of silymarin on the proliferation and glutathione levels of peripheral blood mononuclear cells from beta-thalassemia major patients

Iron toxicity in beta-thalassemia major is the main cause of oxidative stress and cell mediated immune deficiencies. Despite indicative signs of severe oxidative deficiencies associated with beta-thalassemia major, such as decreased level of plasma antioxidants and depletion of erythrocyte glutathione, little is known about intracellular redox status of immune cells. Since glutathione is a primary intracellular antioxidant and plays an essential role in several functions in T cells, in this study intracellular glutathione (GSH) levels as well as proliferation of PHA-activated peripheral blood mononuclear cells (PBMC) were investigated in 28 beta-thalassemia major patients and 28 healthy age-matched individuals. Considering the potential benefits of flavonoids in the therapy of oxidative stress, the effects of silymarin on the GSH levels and proliferation of PBMC from normal and thalassemia individuals were further examined. Quantitative determination of intracellular GSH and proliferative response of PBMC to PHA were performed before and after 72 h incubation of PBMC with various concentrations of silymarin (0, 5, 10, or 20 mug/ml). Results demonstrated a significant reduction of GSH and proliferation in beta-thalassemia major cells; however treatment with silymarin led to restoration of both GSH levels and PBMC proliferation in thalassemia patients. Considerably low levels of GSH and depressed proliferative response of PBMC in beta-thalassemia major may be responsible for the cell mediated immune abnormalities in iron overload conditions. Moreover, the GSH restoration and improvement of PBMC growth by silymarin is a possible explanation for its recently reported antioxidant and immunostimulatory activities. These data suggest the benefit of using flavonoids to normalize immune dysfunction in beta-thalassemia major. The immunomodulatory effects of silymarin in beta-thalassemia major are currently under further investigation in a double blind clinical trial.     

Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles

Engineered nanoparticles (NPs) are widely used in different technologies but their unique properties might also cause adverse health effects. In reviewing recent in vitro and in vivo genotoxicity studies we discuss potential mechanisms of genotoxicity induced by NPs. Various factors that may influence genotoxic response, including physico-chemical properties and experimental conditions, are highlighted. From 4346 articles on NP toxicity, 112 describe genotoxicity studies (94 in vitro, 22 in vivo). The most used assays are the comet assay (58 in vitro, 9 in vivo), the micronucleus assay (31 in vitro, 14 in vivo), the chromosome aberrations test (10 in vitro, 1 in vivo) and the bacterial reverse mutation assay (13 studies). We describe advantages and potential problems with different methods and suggest the need for appropriate methodologies to be used for investigation of genotoxic effects of NPs, in vitro and in vivo.     

Influence of crisis on haemoglobin F level in adult Nigerian sickle cell anaemia patients.

Forty-six Nigerian adult sickle cell anaemia patients were investigated, each in sickle cell crisis and steady state. Forty-three patients had vaso-occlusive crisis while three had haemolytic episodes. Investigations included Packed Cell Volume (PVC), Reticulocyte count and Haemoglobin F estimation. PCV was carried out by the microhaematocrit method while the reticulocytes were counted manually. Haemoglobin F was estimated by the Alkali Denaturation Technique. There was significant anaemia (p < 0.05) and reticulocytosis (p < 0.0001) during the period of crisis compared to the steady state. There was no significant difference (p > 0.05) between HbF level in crisis and that in the steady state. In other words the previously documented increase in HbF during reticulocyte response did not take place in our model. Maybe a 'critical' level of reticulocytosis was not attained. It was also shown that vaso-occlusive crisis did not induce an increase in HbF level suggesting that HbF might be genetically determined at a constant low level throughout life in each of our patients.     

Advancements in Z-DNA: development of inducers and stabilizers for B to Z transition

Z-DNA, an active element in genome, has drawn intense interest in chemical and biological field. Its dynamic and transient state makes it challenging to target and regulate. Thus, stabilizing and inducing Z-DNA both in vitro and in vivo is essential, so far, much many efforts have been made in these aspects. However, Z-DNA's induction and stabilization are always performed in high salt condition and sequence-dependent, limited inducers or stabilizers have been achieved with breakthrough in the aspects of real physiological condition and sequence-independence. Herein, we give a review of some typical kinds of Z-DNA inducers and stabilizers, discussing their inducing or stabilizing condition, mechanism, structural relationship and their limitation as well, attempted to get some implication and guidance for Z-DNA inducer or stabilizer design.     

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.     

Effects of anthracycline derivatives on human leukemia K562 cell growth and differentiation

New derivatives of daunorubicin (DRB), doxorubicin (DOX), and epidoxorubicin (EDOX) with an amidine group bonded to C-3' of daunosamine moiety with either morpholine or hexamethyleneimine ring attached to the amidine group are studied in this paper. We have shown that all of these newly synthesized anthracycline derivatives inhibit human leukemia K562 cell line proliferation but only some of them induce erythroid differentiation when used at subtoxic concentrations. Morpholine derivative of DOX has the greatest potential to inhibit proliferation and to induce differentiation in vitro. The correlation between these two cellular processes was also significant for other tested compounds. In cell cycle analysis, we have demonstrated that those anthracycline derivatives that exert the greatest cytostatic potential caused G(2)/M arrest, which in turn, might contribute to the development of a differentiating phenotype. The concentrations of the compounds used in the study are pharmacologically relevant. These new potent inducers of differentiation might be exploited as anticancer drugs for treatment of leukemia by differentiation therapy.     

Flavonoid antioxidants

In order to ascertain the role of dietary flavonoids as antioxidants in vivo it is necessary to understand the chemical nature of the absorbed forms in the circulation in vivo and how the multiplicity of research findings in vitro reflect the bioactivity of flavonoids in vivo. Only when we gain adequate information on the circulating forms can we begin to understand the targeting to the tissues, whether flavonoids cross the blood-brain barrier, for example, and in what forms. Flavonoids are powerful antioxidants in vitro, but their overall function in vivo has yet to be clarified, whether antioxidant, anti-inflammatory, enzyme inhibitor, enzyme inducer, inhibitor of cell division, or some other role. It should also be emphasised that the reducing properties of flavonoids might contribute to redox regulation in cells, independently of their antioxidant properties, and thus might protect against cell ageing, for example, by working together with the intracellular reductant network. To gain understanding of these issues the factors influencing the absorption of flavonoids in the gastrointestinal tract needs to be established, namely the questions of: de-glycosylation before absorption, conjugation in the small intestine through glucuronidation, sulphation or methylation etc, metabolism and degradation in the colon to smaller phenolic molecules. The forms in which they circulate in vivo will influence their polarity and, thus, their localization and bioactivities in vivo. Finally if antioxidant activities are important, the elucidation of how such properties in vitro relate to the potential for conjugates and metabolites in vivo to act as antioxidants is required. The absorbed flavonoid components might function in the aqueous phase (like vitamin C) or in the lipophilic milieu (as vitamin E) in vivo. This will depend on their polarity properties on uptake, how they are metabolised on absorption, and their resulting structural forms in the circulation.     

Comparison of genotoxicant-modified transcriptomic responses in conventional and epigenetically stabilized primary rat hepatocytes with in vivo rat liver data

The concept of mechanistic toxicogenomics implies that compound-induced changes in gene expression profiles provide valuable information about their mode of action. A growing number of research groups have presented evidence that whole-genome gene expression profiling techniques might be used as tools for in vivo and in vitro generation of gene signatures and elucidation of molecular mechanisms after exposure to toxic compounds. An important issue to be investigated is the in vivo relevance of in vitro-obtained data. In the current study, we compare the gene expression profiles generated in vitro, after exposing conventional and epigenetically stabilized primary rat hepatocytes to well-known genotoxic hepatocarcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene) with those derived in vivo after oral exposure of rats to these compounds. Similar statistical tools were applied on both sets of data. The major molecular pathways affected in the in vivo setting were DNA damage, detoxification and cell survival response, as previously described. In the conventional hepatocyte cultures, two of the three genotoxicants showed quite similar responses as in vivo with respect to these pathways. The third compound (2-nitrofluorene) revealed in vitro response which was not observed in vivo. In the epigenetically stabilized hepatocytes, in contrast to what was expected, the responses were less relevant for the in vivo situation. This study highlights the importance of in vitro/in vivo comparison of data that are generated using in vitro models and shows that conventional primary rat hepatocyte cultures represent an appropriate in vitro model to retrieve mechanistic information on the exposure to genotoxicants.     

A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.     

诱导肿瘤发生免疫原性细胞死亡的疗法研究进展

激活机体的适应性抗肿瘤免疫应答对于长期的抗肿瘤疗效具有重要意义.放射治疗和某些化疗药物等不仅可以诱导细胞凋亡,还可以诱导肿瘤细胞免疫原性细胞死亡(ICD).发生ICD的肿瘤细胞释放损伤相关分子模式(DAMPs),招募抗原呈递细胞吞噬加工肿瘤细胞抗原,并进一步激活T细胞适应性免疫应答.因此,将ICD诱导剂应用到肿瘤治疗中...     

Quantitative drug interactions prediction system (Q-DIPS): a dynamic computer-based method to assist in the choice of clinically relevant in vivo studies

Metabolic drug interactions are a major source of clinical problems, but their investigation during drug development is often incomplete and poorly specific. In vitro studies give very accurate data on the interactions of drugs with selective cytochrome P450 (CYP) isozymes, but their interpretation in the clinical context is difficult. On the other hand, the design of in vivo studies is sometimes poor (choice of prototype substrate, doses, schedule of administration, number of volunteers), with the risk of minimising the real potential for interaction. To link in vitro and in vivo studies, several authors have suggested using extrapolation techniques, based on the comparison of in vitro inhibition data with the active in vivo concentrations of the inhibitor. However, the lack of knowledge of one or several important parameters (role of metabolites, intrahepatocyte accumulation) often limits the possibility for safe and accurate predictions. In consequence, these methods are useful to complement in vitro studies and help design clinically relevant in vivo studies, but they will not totally replace in vivo investigation in the future. We have developed a computerised application, the quantitative drug interactions prediction system (Q-DIPS), to make both qualitative deductions and quantitative predictions on the basis of a database containing updated information on CYP substrates, inhibitors and inducers, as well as pharmacokinetic parameters. We also propose a global approach to drug interactions problems--'good interactions practice--to help design rational drug interaction investigations, sequentially associating in vitro studies, in vitrolin vivo extrapolation and finally well-designed in vivo clinical studies.     

The silence of the genes: epigenetic disturbances in haematopoietic malignancies

Cancer-associated disturbances of regulated DNA methylation include both global hypomethylation and gene-specific (often even cancer-specific) hypermethylation. Both coexist and have become the subject of intense investigation. In haematological neoplasias, distinct sets of genes, including the p15/INK4b cell cycle inhibitor (mostly in myeloid malignancies) as well as p16/INK4a (only very infrequently in myeloid neoplasia), have been well characterised as to incidence of hypermethylation, concurrent gene inactivation and their re-expression following treatment with DNA methylation inhibitors. Several genes frequently methylated in haematological neoplasias have been studied with respect to their prognostic value. With the advance of low-dose schedules of demethylating agents (explored particularly in the elderly patient population) the rationale for reverting the 'hyper-methylator phenotype' has also prompted in vivo studies of gene reactivation following this type of treatment. However, ubiquitous surrogate markers for the efficacy of this type of treatment need to be developed. These may include reactivated haemoglobin F (HbF), as demethylating agents can result in clinically meaningful induction of HbF in patients with haemoglobinopathies. Because 'cancer testis antigens', which provide powerful signals for T cell cytotoxic activity on solid tumour cells, are usually silenced in leukaemia but can be reactivated in vitro and in vivo, they provide a rationale for an immuno-modulatory effect of demethylating therapy.     

Therapeutic modulation of retinoid X receptors – SAR and therapeutic potential of RXR ligands and recent patents

Introduction: Retinoid X receptor (RXR) agonists have a limited role in cancer therapy with bexarotene and alitretinoin as approved drugs but their use is limited by adverse effects. Several evidence from in vitro, in vivo, and small clinical studies points to various further potential applications of RXR ligands in neurodegenerative and inflammatory diseases.

Areas covered: The authors review known RXR ligand classes with their key structure–activity relationships and recent reports on pharmacological effects of RXR modulation. Based on these aspects, the authors evaluate recent patents claiming novel RXR ligands or their use.

Expert opinion: While the use of RXR modulators has been claimed in several novel and promising indications, little progress has been made in the development of innovative rexinoids with improved (subtype-)selectivity. Next-generation RXR modulators that selectively target the RXR subtypes for individual indications may be required to exhaustively exploit the therapeutic potential of RXRs.