Syntheses, Properties, and Aggregation Behavior of Novel Carboxylate-Based Silicone Surfactants.

Two new anionic silicone surfactants were synthesized for the first time from dichloromethylvinylsilane or trichlorovinylsilane through hydrolysis-condensation and then thiol-ene reactions. Their structures were characterized by FT-IR, 1 H NMR and ESI-MS. The surface tension (γ), critical aggregate concentration (CAC), surface pressure at CAC ( Π C A C ${\Pi _{C{\rm{A}}C} }$ ) and minimum surface area per surfactant molecule ( A min ${{A}_{\min } }$ ) were studied by surface tension and electrical conductivity, demonstrating their high surface activity at the gas/liquid interface. Transmission electron microscopy measurements showed that uniform spherical aggregates former in aqueous solution for both surfactants. Moreover, the size of the aggregates was determined to be in the range from 50 to 300 nm by dynamic light scattering.

Demographic Characteristics of Voluntary Donors Registered in Beijing Tongren Hospital Eye Bank of China: A Retrospective Study From 2007 to 2016.

BACKGROUND: Lack of willingness to pledge eyes among the general population is the main cause for the shortage of cornea tissue in China. A few studies have implied that general-population adults with specific demographics showed more willingness to donate their eyes. METHODS: In this study, we analyzed the demographic characteristics of 918 voluntary donors registered in Beijing Tongren Hospital Eye Bank in the past 10 years for possible predictors that might help us to identify potential donors in Beijing and increase the donation rate. All copies of voluntary eye donation application forms filled by the registrants from 2007 to 2016 were collected. Basic demographics listed in the application form were extracted for analysis. Demographics were described as proportions and compared by means of a χ2 test. Besides that, donor counts and proportions of combining 2 demographics from the 4 main demographics were described and compared. RESULTS: Voluntary donors greater than 50 years of age (n = 477, 53.0%) predominated the proportions. Regarding education level and occupation, donors with tertiary education (n = 484, 57.4%) were more numerous than donors with primary and secondary education (n = 355, 42.1%); office clerks, workers, and government officers were more willing to pledge eyes than were donors engaged in other occupations. In addition, donors of the Han race (n = 856, 94.9%) made up the majority and mainly were distributed in 5 central administrative districts of Beijing (n = 629, 77.5%). CONCLUSIONS: The present study suggests that older women (>50 years of age), living in a central district, with higher education level and engaged in white-collar work, were possible predictors for potential donors.

Genetic diversity and population structure of Chinese pony breeds using microsatellite markers.

China is one of the principal origins of ponies in the world. We made a comprehensive analysis of genetic diversity and population structure of Chinese ponies based on 174 animals of five indigenous Chinese pony breeds from five provinces using 13 microsatellite markers. One hundred and forty-four alleles were detected; the mean number of effective alleles among the pony breeds ranged from 5.38 (Guizhou) to 6.78 (Sichuan); the expected heterozygosity ranged from 0.82 (Guizhou) to 0.85 (Debao, Sichuan). Although abundant genetic variation was found, the genetic differentiation was low between the ponies, with 6% total genetic variance among the different breeds. All the pairwise F(ST) values were significant; they varied from 0.0424 for the Sichuan-Yunnan pair to 0.0833 for the Guizhou-Sichuan pair. All five pony breeds deviated from Hardy-Weinberg equilibrium, except the Yunnan pony. Phylogenetic trees of the five pony breeds based on genetic distances were constructed using a neighbor-joining method. The Sichuan and Yunnan ponies were grouped into the same branch, with a high bootstrap support value (97%). Guizhou and Ningqiang ponies were clustered into the same branch with a bootstrap value of 56%, whereas the Debao pony was placed in a separate group, with a bootstrap value of 56%. This grouping pattern was supported by genetic structure analysis.

Hypoxia-ischemia brain damage disrupts brain cholesterol homeostasis in neonatal rats.

PURPOSE: The first 3 weeks of life is the peak time of oligodendrocytes development and also the critical period of cholesterol increasing dramatically in central nervous system in rats. Neonatal hypoxia-ischemia (HI) brain damage happening in this period may disturb the brain cholesterol balance as well as white matter development. MATERIALS AND METHODS: To test this hypothesis, postnatal day 7 (P7) Sprague-Dawley rats were subjected to HI insult. Cholesterol concentrations from brain and plasma were measured. White matter integrity was evaluated by densitometric analysis of myelin basic protein (MBP) immunostaining and electron microscopy. Brain TNF-alpha and IL-6 levels were also measured. RESULTS: HI-induced brain cholesterol, but not the plasma cholesterol, levels decreased significantly during the first three days after HI compared with naïve and sham operated rats (p<0.05). Obvious hypomyelination was indicated by marked reductions in MBP immunostaining on both P10 and P14 (p<0.01) and less and thinner myelinated axons were detected on P21 by electron microscopy observation. High expressions of brain TNF-alpha and IL-6 12 h after HI (p<0.05) were also observed. DISCUSSION: The present work provides evidence that HI insult destroyed brain cholesterol homeostasis, which might be important in the molecular pathology of hypoxic-ischemic white matter injury. Proinflammatory cytokines insulting oligodendrocytes, may cause cholesterol unbalance. Furthermore, specific therapeutic interventions to maintain brain cholesterol balance may be effective for the recovery of white matter function.

"Live" surface ferromagnetism in Fe nanodots/Cu multilayers on Cu(111).

We investigate the crossover behavior from two-dimensional (2D) to three-dimensional in multilayers of magnetic nanodots grown by stacking 2D Fe nanodot assemblies on Cu(111) single crystal substrate with a Cu spacing layer. Using an in situ magneto-optical Kerr effect, we have observed a striking ferromagnetic to spin-glass-like phase transition with an increasing number of Fe dot layers. The topmost layer of the Fe dots survives the phase transition and remains ferromagnetic. This unusual surface ferromagnetism is likely caused by a surface-state-mediated coupling which is stronger than the coupling in bulk layers. This is confirmed by the fact that the critical temperature of the surface ferromagnetism is considerably higher than that of the bulk spin-glass phase in the system.

Construct validity of the Chinese version of the disabilities of the arm, shoulder and hand questionnaire (DASH-HKPWH).

The study evaluated the construct validity of the Chinese Hong Kong version of the disabilities of the arm, shoulder and hand questionnaire (DASH-HKPWH). Three hundred and thirty four patients with a broad range of upper extremity disorders were recruited into the study. Each completed DASH-HKPWH and SF-36 forms and their pain intensity (numeric pain rating scale) and grip strength were also measured. The mean DASH-HKPWH score was 40 (SD=20). Principal component factor analysis identified a single factor, which accounted for 47% of the total variance. Pearson correlation was applied to assess convergent and divergent validity of the DASH-HKPWH by comparison with the above-mentioned subjective and objective measurements. Our findings were comparable to the DASH of other languages.

An evaluation of the P450 inhibition and induction potential of daptomycin in primary human hepatocytes.

Two in vitro studies assessed the potential of daptomycin (Cubicin), a newly marketed antibiotic, to affect the cytochrome P450 (CYP450) isoforms in primary cultured human hepatocytes. Both induction and inhibition of isoforms 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were evaluated. The highest concentrations of daptomycin used in both the induction and inhibition assays were approximately eight-fold higher than the peak total drug concentration (50-60 microg/mL), or the peak free drug concentration (estimated 5-6 microg/mL), in plasma at the clinical dose regimen of 4 mg/kg qd. Results in primary human hepatocytes indicate that daptomycin, at concentrations up to 400 microg total drug/mL, demonstrated no biologically significant induction of any of the CYP450 isoform activities in comparison with the negative control or known inducers. At daptomycin concentrations up to 40 microg free drug/mL, no biologically significant inhibition of the activities of these CYP450 isoforms was observed as compared with known inhibitors. The human hepatocyte results demonstrate that daptomycin has no effects on hepatic CYP450-mediated drug metabolism and, therefore, suggest that daptomycin is unlikely to show potential for pharmacokinetic interactions with concomitantly administered drugs that are metabolized by CYP450 isoforms.

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation.

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.

Species comparison in P450 induction: effects of dexamethasone, omeprazole, and rifampin on P450 isoforms 1A and 3A in primary cultured hepatocytes from man, Sprague-Dawley rat, minipig, and beagle dog.

Induction of P450 isoforms 1A (CYP1A) and 3A (CYP3A) by model inducers dexamethasone, omeprazole and rifampin was evaluated in primary cultured hepatocytes from man and laboratory animals. Inducer-specific species-differences were observed. Results with human hepatocytes from six human donors consistently show that both rifampin and dexamethasone were inducers of CYP3A activity (measured as testosterone 6beta-hydroxylase activity), with rifampin being more potent. Conversely, in rat hepatocytes, dexamethasone was a potent CYP3A inducer while rifampin was not an inducer. Rifampin but not dexamethasone induced CYP3A in minipig and beagle dog hepatocytes. Omeprazole was a potent inducer of CYP1A activity (measured as ethoxyresorufin-O-deethylase activity) in human, beagle dog and minipig hepatocytes, and not an inducer in rat hepatocytes. The species-differences observed suggest that human hepatocytes represent the most appropriate preclinical experimental system for the evaluation of P450 induction in human.

Screening for human ADME/Tox drug properties in drug discovery.

There is no doubt that ADME/Tox drug properties, absorption, distribution, metabolism, elimination and toxicity, are properties crucial to the final clinical success of a drug candidate. It has been estimated that nearly 50% of drugs fail because of unacceptable efficacy, which includes poor bioavailability as a result of ineffective intestinal absorption and undesirable metabolic stability(1). It has also been estimated that up to 40% of drug candidates have failed in the past because of safety issues(2). In this review, the methodologies that are available for use in drug development as in vitro human-based screens for ADME/Tox drug properties are discussed.

Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes.

We studied the effects of acetonitrile, dimethyl sulfoxide (DMSO), and methanol (MeOH) in human hepatocytes on cytochrome P450 (CYP) and phase II conjugation activities: phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), testosterone 6beta-hydroxylation (CYP3A4), and umbelliferone glucuronidation and sulfation. The solvents were evaluated at concentrations (v/v) of 0.1, 1, and 2%. Previously cryopreserved human hepatocytes pooled from multiple donors were used as suspension cultures in this study. DMSO was found to inhibit CYP2C9 and CYP2C19, CYP2E1, and CYP3A4 in a concentration-dependent manner. At 2% DMSO, the activities for the four isoforms were approximately 40% (CYP2C9), 23% (CYP2C19), and 11% (CYP2E1) of that observed for 0.1% acetonitrile and 45% (CYP3A4) of that observed for 1% acetonitrile. No apparent inhibitory effects were observed for the other activities evaluated. Methanol was found to inhibit CYP2C9 and CYP2E1 activities, but to a lesser extent than DMSO. Acetonitrile had no apparent effects on any of the on any of the activities evaluated. These findings should be considered when choosing an organic solvent for metabolism studies with human hepatocytes.

In vitro human tissue models in risk assessment: report of a consensus-building workshop.

Advances in the technology of human cell and tissue culture and the increasing availability of human tissue for laboratory studies have led to the increased use of in vitro human tissue models in toxicology and pharmacodynamics studies and in quantitative modeling of metabolism, pharmacokinetic behavior, and transport. In recognition of the potential importance of such models in toxicological risk assessment, the Society of Toxicology sponsored a workshop to evaluate the current status of human cell and tissue models and to develop consensus recommendations on the use of such models to improve the scientific basis of risk assessment. This report summarizes the evaluation by invited experts and workshop attendees of the current status of such models for prediction of human metabolism and identification of drug-drug interactions, prediction of human toxicities, and quantitative modeling of pharmacokinetic and pharmaco-toxicodynamic behavior. Consensus recommendations for the application and improvement of current models are presented.

2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes.

We report here a novel observation that 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) induced predominantly cytochrome P4501A1 (CYP1A1) in rat hepatocytes and predominantly CYP1A2 in human hepatocytes. As part of our research program to evaluate species-differences in response to CYP inducers, we studied the effects of TCDD on CYP1A activity, protein, and gene expression in primary cultures of rat and human hepatocytes. TCDD was found to induce CYP1A activity, measured as ethoxyresorufin-O-deethylase (EROD) activity, in both rat and human hepatocytes. TCDD induction of EROD activity in human hepatocytes (2-5 fold of concurrent solvent control), was significantly lower than that found in rat hepatocytes ( 20-fold of concurrent solvent control). Two structural analogs of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF), were also evaluated. As observed for TCDD, human hepatocytes consistently showed a lower response than rat hepatocytes. As most TCDD-related effects are believed to be mediated via binding of the TCDD-Ah receptor (AhR) complex to DNA, nuclear AhR levels were measured in rat and human hepatocytes after TCDD treatment. We found that the nuclear AhR levels in TCDD-treated rat hepatocytes were approximately 4 times higher than found in TCDD-treated human hepatocytes. However, the estimated binding affinity of [3H]TCDD to nuclear AhR from rat hepatocytes was similar. The species difference in response to TCDD was further evaluated by analysis of CYP1A1 and CYP1A2 mRNA levels using Northern analysis, and P4501A1 and 1A2 protein levels using Western immunoblotting. Results showed that, at both gene expression and protein levels, TCDD induced predominantly CYP1A1 in rat hepatocytes and CYP1A2 in human hepatocytes.

Effects of cytochrome P450 inducers on 17alpha-ethinyloestradiol (EE2) conjugation by primary human hepatocytes.

AIMS: Our objective was to elucidate further the underlying mechanism responsible for therapeutic failures observed with concomitant administration of the oral contraceptive 17alpha-ethinyloestradiol (EE2 ) and rifampicin. METHODS: We investigated both oxidative and direct conjugative [3H]-EE2 metabolism by human liver S9 fraction and the effect of known enzyme-inducing drugs using a human hepatocyte induction model in vitro. RESULTS: Cofactor dependent [3H]-EE2 metabolism by human liver S9 fraction produced 2-hydroxy-[3H]-EE2, 2-methoxy-[3H]-EE2, and direct [3H]-EE2 sulphate and glucuronide conjugates. Only two detectable metabolites of [3H]-EE2 were produced by the S9 fraction in the presence of all cofactors: [3H]-EE2-3-sulphate (75.7+/-7.6% s. d.) and 2-methoxy-3H-EE2 (2.6%+/-0.5% s.d.). Human hepatocytes extensively metabolized [3H]-EE2 to its glucuronide and sulphate conjugates. Small amounts of a 2-methoxy-[3H]-EE2 3-conjugate, < or = 10%, was observed but no. 2-hydroxy-[3H]-EE2 was detected. An unexpected finding in our study was increased [3H]-EE2-3-sulphate production (1.5-3.3 fold, n=3 donor livers) by hepatocytes pretreated with rifampicin compared to control hepatocytes. No statistically significant increase in [3H]-EE2-3-sulphation was observed in hepatocytes pretreated with 3-methylcholanthrene, phenobarbitone, dexamethasone, or omeprazole over nontreated hepatocytes. To our knowledge, this is the first observation of sulphotransferase induction by rifampicin in human hepatocytes in vitro resulting in increased [3H]-EE2 sulphation. CONCLUSIONS: Our data indicate that the major EE2 metabolic products formed by human hepatocytes in vitro are direct EE2 conjugates with EE2 oxidation representing minor pathways. Further studies are required to establish the mechanism of sulphotransferase induction and the clinical relevance of our findings.

Overview: hepatocytes and cryopreservation--a personal historical perspective.

Hepatocytes represent an important tool for the investigation of species differences in drug metabolism and toxicity. Data obtained with hepatocytes from multiple animal species, including man, allow better prediction of the effects of xenobiotics in man. Cryopreservation of hepatocytes extends the use of this important experimental system by enhancing the convenience of its use. Also, it allows the researchers to perform experiments not plausible with freshly isolated hepatocytes, such as the direct comparison of xenobiotic toxicity and metabolism in hepatocytes from multiple human donors in a single experiment.

Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential.

Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential.

Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel.

Successful cryopreservation of freshly isolated hepatocytes would significantly decrease the need for freshly-procured livers for the preparation of hepatocytes for experimentation. Hepatocytes can be prepared, cryopreserved, and used for experimentation as needed at different times after isolation. Cryopreservation is especially important for research with human hepatocytes because of the limited availability of fresh human livers. Based on the cumulative experience of this international expert panel, a consensus was reached on the various aspects of hepatocyte cryopreservation, including cryopreservation and thawingprocedures and applications of the cryopreserved hepatocytes. Key to successful cryopreservation includes slow addition of cryopreservants, controlled-rate freezing with adjustment for the heat of crystallization, storage at -150 degrees C, and rapid thawing. There is a general consensus that cryopreserved hepatocytes are useful for short-term xenobiotic metabolism and cytotoxicity evaluation.

An evaluation of the cytochrome P450 induction potential of pantoprazole in primary human hepatocytes.

Primary human hepatocytes contain a full complement of human drug-metabolizing enzymes and therefore represent a relevant experimental system for the evaluation of pharmacokinetic drug-drug interaction potential in human. In this study, the cytochrome P450 (CYP) induction potential of pantoprazole (PAN) was evaluated and compared to two other proton pump inhibitors (PPIs), omeprazole (OM) and lansoprazole (LAN). Primary human hepatocytes from three donors were studied. The hepatocytes were cultured for 3 days, followed by treatment for 3 days with the PPIs at 2, 5 and 10 microM. Two other known CYP inducers, 3-methylcholanthrene at 1 microM and rifampin at 50 microM, were also evaluated. Induction potentials of these chemicals for CYP1A and CYP3A were evaluated by isozyme activity and isozyme content. 7-Ethoxyresorufin-O-deethylase and testosterone 6beta-hydroxylase activities were used as endpoints for CYP1A and CYP3A, respectively. Isozyme protein contents of CYP1A and CYP3A were evaluated via Western blotting. The results showed that for CYP1A induction, the rank ordering in induction potential was consistently OM > LAN > PAN. CYP3A induction by the PPI's were observed in two of the three hepatocyte cultures, with no apparent differences in induction potency for the three compounds. Our results on CYP1A induction suggest that PAN has a lower drug-drug interaction potential than OM and LAN.

Estrogen sulfotransferase expression in the human liver: marked interindividual variation and lack of gender specificity.

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation of estrogen at the 3'-hydroxyl position using 3'-phosphoadenosine-5'-phosphosulfate as an activated sulfate donor. Sulfonation renders the hormone biologically inactive as well as changing its half-life within the human body. Studies in the rat and mouse have suggested that expression of EST in the liver is age- and sex-dependent, being prominent only in sexually mature young males. Although a human EST cDNA has previously been cloned, the characteristics of hepatic EST expression in human subjects remain to be defined. In this study, we have investigated and compared the expression of EST in 10 human liver samples by using an EST-specific antibody and performing enzyme activity assays. We found a marked interindividual variation (up to 25-fold) in the hepatic expression of EST. However, EST protein level in the human liver is correlated neither with gender nor with age. Interestingly, paired-group analysis revealed a statistically significant difference in the hepatic expression of EST protein and activity between alcohol users and nonusers. We conclude that, unlike what is observed in the rodent liver, EST expression in the human liver is not sex-limited. Thus hepatic EST may play a role in estrogen metabolism and homeostasis in both genders of human subjects. The marked individual variation suggests that EST gene expression is subject to sensitive control by genetic or environmental factors. The potential correlation between alcohol consumption and hepatic EST expression deserves further evaluation.