Can histamine cause an enhancement of the cellular uptake and cytotoxicity of doxorubicin-loaded polylactide nanoparticles?

Histamine (His) in humans is physiologically involved in neurotransmission and increases vascular permeability during the development of inflammatory response and immunity. It could be used to enhance drug-loaded nanoparticles (NPs) distribution. However, it cannot be freely delivered due to the risk of His-dose-dependent deleterious effects. His can be attached to the polymeric backbone during polymerization to overcome this limitation. In this study, His was used as an initiator of lactide polymerization, and the obtained macromolecules were subsequently used to prepare doxorubicin (DOX)-loaded NPs by nanoprecipitation and microfluidics for examination of anti-cancer properties. Notably, the in vitro activity towards gastric cancer cells (AGS) of the NPs composed of histamine-functionalized polylactides (PLAs) was greatly enhanced compared to control NPs built from hydroxy­functionalized PLAs. Furthermore, Zonula occludens-1 (ZO-1) tight junction protein production was significantly diminished after treating cells with DOX-loaded NPs assembled with PLAs with histamine residues. These results demonstrate the synergistic effect in cytotoxicity towards gastric cancer cells of DOX and the histamine that are carried by NPs. It is believed that His-DOX NPs strategy may lead to effective, targeted, and low-toxic delivery of drugs into cancer cells.

pH-tunable nanoparticles composed of copolymers of lactide and allyl-glycidyl ether with various functionalities for the efficient delivery of anti-cancer drugs.

The designing of biocompatible nanocarriers for the efficient delivery of their cargos to the desired targets remains a challenge. In this regard, the most promising strategy relies on the construction of pH- or thermo-responsive nanoparticles (NPs). However, it is also important to preserve the balance between the responsiveness of the carrier and their stability in physiological conditions. Therefore, we described a new family of copolymers of lactide and allyl-glycidyl ether which were subsequently modified by thiol-ene reaction to functionalize the resulting copolymer with acetylcysteine (ACC) or thioglycolic acid (tGA) moieties. Subsequently, these copolymers were used to obtain blank and doxorubicin (DOX) loaded NPs with an average diameter of about 50-100 nm. Interestingly, the NPs were stable in different pH conditions, however, the presence of ACC or tGA units in the polymeric chain allows for the reduction of the undesired burst release due to the supramolecular interactions between polymeric pedant groups and DOX. The release tests of DOX from NPs showed that DOX release rate decrease depending on the pH values and the copolymer functionalization in order of non-modified NPs > ACC-modified NPs > tGA functionalized NPs. Most importantly, the MTT assay showed that all blank NPs are non-toxic against the normal L929 cell line. Subsequently, the antitumor efficiency of the obtained NPs was tested towards L929 (murine fibroblast cell line), HeLa (cervical cancer), and AGS (human gastric adenocarcinoma cancer) cells. The results demonstrated that DOX-loaded NPs efficiently induce the reduction in the viability of the HeLa and AGS cell, and this reduction in the viability was even below 20 % for the AGS cells. Together with their biocompatibility, the obtained NPs offer a novel route for the preparation of nanocarriers for the controlled and efficient delivery of anticancer drugs.

Stereocomplexed PLA microspheres: Control over morphology, drug encapsulation and anticancer activity.

Lung cancer is the leading cause of cancer death because of smoking and air pollution. Therefore, new ideas should be provided for lung cancer treatment in which the delivery of anticancer drugs to the local tumor site can be achieved. For this purpose, we propose the use of stereocomplexed spherical microspheres with sizes between 0.5 and 10 µm loaded with doxorubicin (DOX) to be administered through the nasal route. In order to gain control over the microsphere morphology, size, and drug loading capacity, we systematically studied the influence of the solvent used for preparation and the functionalization of their building blocks, namely poly-l-lactide (PLLA) and poly-d-lactide (PDLA) with blocked or unblocked l-proline moieties. We could demonstrate that DOX release is generally determined by the size of the microspheres. The antiproliferative activity of DOX released from the different microspheres was shown in vitro using the A549 lung cancer cell line as a model. Moreover, when in direct contact to the cancer cells, smaller microspheres were uptaken and could serve as a reservoir for local drug release. Our findings not only provide a novel strategy to prepare PLA microspheres with controllable morphology and release of anti-cancer drugs but also offer additional possibilities for the application of stereocomplexed particles in anticancer therapy, with suitable sizes for nasal administration.

Self-Assembly of Triblock Copolymers from Cyclic Esters as a Tool for Tuning Their Particle Morphology.

This paper presents the effect of end groups, chain structure, and stereocomplexation on the microparticle and nanoparticle morphology and thermal properties of the supramolecular triblock copolyesters. Therefore, the series of the triblock copolymers composed of l,l- and d,d-lactide, trimethylene carbonate (TMC), and ε-caprolactone (CL) with isopropyl ( iPr) or 2-ureido-4-[1 H]-pyrimidinone (UPy) end groups at both chain ends were synthesized. In addition, these copolymers were intermoleculary stereocomplexed by polylactide (PLA) blocks with an opposite configuration of repeating units to promote their self-assembly in various organic solvents. The combination of two noncovalent interactions of the end groups and PLA enantiomeric chains leads to stronger interactions between macromolecules and allows for alteration of their segmental mobility. The simple tuning of the copolymer microstructure and functionality induced the self-assembly of macromolecules at liquid/liquid interfaces, which consequently leads to their phase separation in the form of particles with diameters ranging from 0.1 µm to 10 µm. This control is essential for their potential applications in the biomedical field, where biocompatible and well-defined microparticles and nanoparticles are highly desirable.

Thromboelastographic profile of goat blood after the experimental injury of the femoral artery and use of QuikClot gauze and Celox gauze dressings.

The aim of this study was to evaluate the suitability of thromboelastometry for the analysis of blood test results in goats after the use of hemostatic dressings to control massive bleeding. The study was carried out on 12 goats, 6 animals in each of two subgroups. In all experimental animals incision of the femoral artery was performed, and bleeding was controlled with QuikClot gauze in the first group and Celox gauze in the second group. Dressings were applied for 60 minutes. Blood samples for thromboelastometry were collected from the jugular vein before the incision and 60 min after the application of a dressing. Clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF) and α angle (°) were measured in three standard ROTEM assays (system with generation of reaction curve, numerical parameters and size of the blood clot): intrinsic coagulation pathway (INTEM), extrinsic coagulation pathway (EXTEM) and functional fibrinogen (FIBTEM). Complete hemostasis of the injured femoral artery was found in all goats. No significant differences between pre- and post-incision thromboelastometric parameters were found in any tests in any of the groups, which indicates that the use of dressings was not associated with blood coagulation disorders. This study is the first to describe the use of thromboelastometry in goats for the assessment of clot formation and hemostatic disorders.

The New 3D Printed Left Atrial Appendage Closure with a Novel Holdfast Device: A Pre-Clinical Feasibility Animal Study.

INTRODUCTION: Many patients undergoing cardiac surgery have risk factors for both atrial fibrillation (AF) and stroke. The left atrial appendage (LAA) is the primary site for thrombi formation. The most severe complication of emboli derived from LAA is stroke, which is associated with a 12-month mortality rate of 38% and a 12-month recurrence rate of 17%. The most common form of treatment for atrial fibrillation and stroke prevention is the pharmacological therapy with anticoagulants. Nonetheless this form of therapy is associated with high risk of major bleeding. Therefore LAA occlusion devices should be tested for their ability to reduce future cerebral ischemic events in patients with high-risk of haemorrhage. AIM: The aim of this study was to evaluate the safety and feasibility of a novel left atrial appendage exclusion device with a minimally invasive introducer in a swine model. MATERIALS AND METHODS: A completely novel LAA device, which is composed of two tubes connected together using a specially created bail, was designed using finite element modelling (FEM) to obtain an optimal support force of 36 N at the closure line. The monolithic form of the occluder was obtained by using additive manufacturing of granular PA2200 powder with the technology of selective laser sintering (SLS). Fifteen swine were included in the feasibility tests, with 10 animals undergoing fourteen days of follow-up and 5 animals undergoing long-term observation of 3 months. For one animal, the follow-up was further prolonged to 6 months. The device was placed via minithoracotomy. After the observation period, all of the animals were euthanized, and their hearts were tested for LAA closure and local inflammatory and tissue response. RESULTS: After the defined observation period, all fifteen hearts were explanted. In all cases the full closure of the LAA was achieved. The macroscopic and microscopic evaluation of the explanted hearts showed that all devices were securely integrated in the surrounding tissues. No pericarditis or macroscopic signs of inflammation at the site of the device were found. All pigs were in good condition with normal weight gain and no other clinical symptoms. CONCLUSION: This novel 3D printed left atrial appendage closure technique with a novel holdfast device was proven to be safe and feasible in all pigs. A benign healing process without inflammation and damage to the surrounding structures or evidence of new thrombi formation was observed. Moreover, the uncomplicated survival and full LAA exclusion in all animals demonstrate the efficacy of this novel and relatively cheap device. Further clinical evaluation and implementation studies should be performed to introduce this new technology into clinical practice.

[Congenital prepubic sinus: case report]. / Seno prepúbico congénito. A propósito de un caso.

The prepubic congenital sinus is a rare type of urethral duplication of unknown origin. Different embryological theories and classifications has been proposed. Most cases are diagnosed during childhood. The Retrograde Urethrocystography is very important as it determines the diagnosis and helps choosing the best treatment option. We present a case of a prepubic congenital sinus in a 39 years old male who presented with purulent discharge from an accessory meatus in the base of the penis.

High-latitude controls of thermocline nutrients and low latitude biological productivity.

The ocean's biological pump strips nutrients out of the surface waters and exports them into the thermocline and deep waters. If there were no return path of nutrients from deep waters, the biological pump would eventually deplete the surface waters and thermocline of nutrients; surface biological productivity would plummet. Here we make use of the combined distributions of silicic acid and nitrate to trace the main nutrient return path from deep waters by upwelling in the Southern Ocean and subsequent entrainment into subantarctic mode water. We show that the subantarctic mode water, which spreads throughout the entire Southern Hemisphere and North Atlantic Ocean, is the main source of nutrients for the thermocline. We also find that an additional return path exists in the northwest corner of the Pacific Ocean, where enhanced vertical mixing, perhaps driven by tides, brings abyssal nutrients to the surface and supplies them to the thermocline of the North Pacific. Our analysis has important implications for our understanding of large-scale controls on the nature and magnitude of low-latitude biological productivity and its sensitivity to climate change.

Atomic force microscopy study of living diatoms in ambient conditions.

We present the first in vivo study of diatoms using atomic force microscopy (AFM). Three chain-forming, benthic freshwater species -Eunotia sudetica, Navicula seminulum and a yet unidentified species - are directly imaged while growing on glass slides. Using the AFM, we imaged the topography of the diatom frustules at the nanometre range scale and we determined the thickness of the organic case enveloping the siliceous skeleton of the cell (10 nm). Imaging proved to be stable for several hours, thereby offering the possibility to study long-term dynamic changes, such as biomineralization or cell movement, as they occur. We also focused on the natural adhesives produced by these unicellular organisms to adhere to other cells or the substratum. Most man-made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. Diatoms produce adhesives that are extremely strong and robust both in fresh- and in seawater environments. Our phase-imaging and force-pulling experiments reveal the characteristics of these natural adhesives that might be of use in designing man-made analogues that function in wet environments. Engineering stable underwater adhesives currently poses a major technical challenge.

A novel fluorescent silica tracer for biological silicification studies.

BACKGROUND: Biological silica production has drawn intense attention and several molecules involved in biosilicification have been identified. Cellular mechanisms, however, remain unknown mainly due to the lack of probes required for obtaining information on live specimens. RESULTS: The fluorescence spectra of the compound 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole (PDMPO) are affected by the presence of >3.2 mM silicic acid. Increase in intensity and shift in the fluorescence coincide with the polymerization of Si. The unique PDMPO-silica fluorescence is explored here to visualize Si deposition in living diatoms. The fluorophore is selectively incorporated and co-deposited with Si into the newly synthesized frustules (the outer silica shells) showing an intense green fluorescence. CONCLUSIONS: We suggest that a fluorescence shift is due to an interaction between PDMPO and polymeric silicic acid. PDMPO is an excellent probe for imaging newly deposited silica in living cells and has also a potential for a wide range of applications in various Si-related disciplines, including biology of living organisms as diatoms, sponges, and higher plants, clinical research (e.g. lung fibrosis and cancer, bone development, artificial bone implantation), and chemistry and physics of materials research.

[Should we operate goiter in the elderly?]. / Czy operowac wole u chorych w wieku podeszlym?

The aim of this study was to compare the elderly patients group to the group of others undergoing surgical treatment due to goitre regarding type of goitre, extent of surgical procedures and postoperative follow-up (including early postoperative complications). 5872 patients with various type of goitre (between 1984 and 1998) were surgically treated, among them were 5244 (89.3%) females and 628 (10.7%) males (sex ratio as 8.1:1). The mean age was 46.1 (10 to 95 years). 278 (4.7%) patients were above 70 years of age (235-84.5% females and 43-15.5% males), their mean age was 73.7 years. The increase of malignant goitre was evident among the elderly patients (19.9% vs 5.5%). The dominant types were anaplastic cancers and malignant non-Hodgkin lymphomas. Much more frequently a giant goitre was diagnosed (20% vs 6.1%). It was localised substernally (39.6%) or intrathoracic (4.7%). The elderly prepared properly underwent surgical treatment quite well. Among early postoperative complications in the elderly dominant were surgical ones (6.8%), most frequently the injuries of the recurrent laryngeal nerve (4.67% vs 1.14%). Perioperative mortality regarded mostly the elderly with disseminated anaplastic cancer (3.6% vs. 0.2%). Surgical treatment of goitre in the elderly is a safe and justified method. High incidence of malignant tumours especially anaplastic cancers among the elderly should encourage to operation as early as possible.

Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression.

Incorporation of a central polypurine tract (cPPT) and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression. We compared the effects of these elements individually and together on transduction efficiency and gene expression, using lentivirus vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) and encoding enhanced green fluorescent protein (GFP) and rat erythropoietin (EPO). The transduction efficiency was greater than 2-fold higher in the vector containing the PRE element, 3-fold higher in vector encoding the cPPT element, and 5-fold increased in the GFP virus containing both cPPT and PRE elements relative to the parent virus. In comparison with parent vector the mean fluorescence intensity (MFI) of GFP expression was 7-fold higher in cells transduced with virus containing PRE, 6-fold increased in cells transduced with virus containing cPPT, and 42-fold increased in GFP-virus containing both cPPT and PRE elements. EPO-virus containing a PRE element showed a nearly 5-fold increase in EPO secretion over the parent vector, and the vector encoding both PRE and cPPT showed a 65-fold increase. Thus, lentivirus vectors incorporating both PRE and cPPT showed expression levels significantly increased over the sum of the components alone, suggesting a synergistic effect.

Micromechanical and structural properties of a pennate diatom investigated by atomic force microscopy.

The mechanisms behind natural nanofabrication of highly structured silicas are increasingly being investigated. We have explored the use of a standard Nanoscope III Multimode atomic force microscope (AFM) to study the silica shell of diatoms. The delicate structures of the shell surface of the diatom Navicula pelliculosa (Bréb.) Hilse were imaged and the shell's micromechanical properties were measured semi-quantitatively with a resolution down to approximately 10 nm. The technique to measure elasticity and hardness with the AFM was demonstrated to be useable even on these hard glass-like surfaces. Different experimental configurations and evaluation methods were tested. They gave a consistent result of the shell micromechanical properties. The first results showed that the diatom shell's overall hardness and elasticity was similar to that of known silicas. However, regions with different mechanical properties were distinguished. The elastic modulus varied from 7 to 20 GPa, from 20 to 100 GPa and from 30 to hundreds of GPa depending on the location. In general, the hardness measurements showed similar spatial differences. The hardness values ranged from 1 to 12 GPa but one specific part of the shell was even harder. Hence, certain localized regions of the shell were significantly harder or more elastic. These regions coincide with known characteristic features and mechanisms appearing at the different stages of the shell's growth. These results show that this method serves as a complementary tool in the study of silica biomineralization, and can detect eventual crystalline phases.

Atrial fibrillation after coronary artery bypass grafting: does the type of procedure influence the early postoperative incidence?

OBJECTIVE: Atrial fibrillation (AF), the common postoperative complication, has been observed after coronary artery bypass grafting (CABG) in 7--40% of patients. Cardiopulmonary bypass (CPB), eliminated in off-pump operations (OPCABG) may decrease the incidence of AF, whereas the combination of CABG with heart valve replacement may result in more frequent postoperative atrial fibrillation. The aim of our study was to compare the early postoperative AF incidence rate during ICU stay in three groups of patients: after CABG, OPCABG, and CABG combined with valve replacement. MATERIAL AND METHODS: A prospective study of 906 consecutive patients was carried out between January 1999 and January 2000. Clinical profile of 906 patients, including factors having potential influence on postoperative AF did not showed any significant differences between the groups. The presence of arrhythmia history was the reason of excluding 85 patients from the statistical analysis. The observation was performed in each case during ICU-stay, using a HP system for continuous automated arrhythmia analysis. Early postoperative incidence of AF was recorded and compared between three groups of patients: 650 after conventional CABG, 118 after OPCABG, and 53 after CABG combined with valve replacement. Chi-square and a Mann--Whitney tests, Statistica 5.0 PL were used for the statistical analysis. RESULTS: Atrial fibrillation occurred during the postoperative ICU stay in 9.8% of patients after CABG, in 10.2% after OPCABG, and in 21% after CABG combined with valve replacement. There was no significant difference between CABG and OPCABG groups (P=0.965). The confidence interval of the odds ratio ranges from 0.5 to 1.85. Consequently, an increased risk would be possible for both methods. We observed a statistically significant increase of the early postoperative atrial fibrillation incidence rate in patients after CABG combined with valve replacement, when compared with both CABG + OPCABG groups (P=0.005). CONCLUSIONS: (1) Atrial fibrillation is a common postoperative complication after myocardial revascularization procedures which prolongs ICU stay. (2) The study did not show that the incidence of postoperative AF is influenced by the technique of coronary artery bypass grafting: with or without CPB. (3) The prevalence of postoperative AF increase when CABG is combined with valve replacement.

Evaluation of sequence variation and selection in the bindin locus of the red sea urchin, Strongylocentrotus franciscanus.

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5' end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5' region of S. franciscanus bindin is a result of neutral evolution.

Genetic structure of populations of the red sea urchin, Strongylocentrotus franciscanus.

Population subdivision was evaluated in the red sea urchin, Strongylocentrotus franciscanus, using DNA sequence data from 134 adult individuals collected in 1995 and 1996. On average 22 individuals were sequenced from six geographic locations between Alaska and Baja California (N=134), nearly the full extent of the species range. DNA sequence data was obtained from direct sequencing of a 273 base pair region of the bindin gene, which encodes a sperm fertilization protein. Results indicate that bindin is sufficiently polymorphic to serve as a genetic marker. We identified 14 unique alleles present in the entire range sampled with a maximum of eight alleles at a specific site. Mean pairwise comparison of the 14 unique alleles indicates moderate sequence diversity (p-distance=1.06). Although there is conflicting evidence to suggest that Alaska populations may deviate from the Hardy-Weinberg expectations, analysis of bindin genotype frequencies indicate that it is not possible to reject the null hypothesis of random mating throughout the species range. The results of a chi-square test with pooling conform to Hardy-Weinberg expectations for all populations (P>0.05) except for the Alaska population (P=0.037). Inbreeding coefficients are consistent with this result and suggest that for the bindin locus, there is high gene flow. These results are compared with previously published results of genetic substructuring in sea urchins to examine relationships among population structure, dispersal potential and biogeography.

Patterns of CYP26 expression in human prenatal cephalic and hepatic tissues indicate an important role during early brain development.

CYP26 (P450RAI) catalyzes catabolic retinoic acid (RA) hydroxylation and thereby appears to play a critical role in retinoid signaling pathways during development. In this study, a quantitative competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for evaluation of CYP26 message levels in human prenatal tissues. Statistical analyses of transcription levels in 12 prenatal human brains and six prenatal human livers demonstrated good sensitivity and reproducibility. Quantitative profiles of CYP26 gene expression in early (gestational days 57-110) prenatal cephalic and hepatic tissues and comparisons with adult counterparts are reported for the first time. Prenatal cephalic tissues at days 57-67 exhibited values of 1950+/-420 (CYP26 molecules/10(6) GAPDH molecules) whereas prenatal cephalic tissues at days 105-110 exhibited values of 22300+/-4450 (CYP26 molecules/10(6) GAPDH molecules), indicating a sharp developmental increase (approximately 11-fold). Levels in human adult cephalic tissues were slightly less than the prenatal cephalic levels measured during the earliest stages of gestation and were approximately 3-fold lower than those measured in adult human hepatic tissues. Levels in human prenatal hepatic tissues at days 63-110 gestation were less than 800 (CYP26 molecules/10(6) GAPDH molecules) and did not exhibit developmental increases. Considered together, the data have strong implications for the importance of CYP26 in early development of the human brain.

Catalysis of drug oxidation during embryogenesis in human hepatic tissues using imipramine as a model substrate.

We investigated the catalysis of drug monooxygenation by human embryonic hepatic tissues at a very early stage of gestation (days 52-59). Imipramine was used as a model substrate and the metabolites generated were identified and quantified by electrospray mass spectroscopy and HPLC. The primary metabolite generated was desipramine. It was reported previously from this and other laboratories that cytochrome P-450 monooxygenase (CYP) 1A1, 1B1, 2E1, and 3A7 are each expressed in human embryonic hepatic tissues, and selective inhibitors were therefore used to elucidate their respective roles. Furafylline did not inhibit the reaction, supporting that CYP1A2 was not expressed in human embryonic hepatic tissues. Diethyldithiocarbamate also failed to inhibit the same reaction, suggesting that CYP2E1 did not play a significant role in catalyzing the reaction. Triacetyloleandomycin inhibited the reaction by approximately 90%, suggesting that CYP3A7 was primarily responsible for catalyzing the reaction. However, alpha-naphthoflavone inhibited the same reaction by approximately 70%, suggesting that CYP1A1 and/or CYP1B1 may also catalyze the reaction substantially. To explore this issue more, a cDNA-expressed human CYP3A7 (CYP3A7 SUPERSOMES) was incubated with alpha-naphthoflavone (1 microM). Generation of desipramine was inhibited by approximately 40 to 50%. The addition of the CYP3A subfamily selective inhibitor triacetyloleandomycin (1 microM) produced no statistically significant inhibition in reactions catalyzed by CYP1A1 or 1B1 SUPERSOMES. Taken together, the results indicated that CYP3A7 was the major if not sole isoform responsible for catalysis of the N-demethylation of imipramine in human hepatic tissues during embryogenesis.

Catalytic activity and quantitation of cytochrome P-450 2E1 in prenatal human brain.

Cytochrome P-450 2E1 (CYP2E1) is a readily inducible hemoprotein that catalyzes the oxidation of endogenous compounds and many low molecular weight xenobiotics. As the major component of the microsomal ethanol oxidizing system, it contributes significantly to ethanol metabolism and the formation of the highly reactive metabolite acetaldehyde. The leaky property of this enzyme results in the generation of reactive oxygen species that can induce oxidative stress and cytotoxic conditions deleterious to development. To further investigate the proposed role of CYP2E1 in the etiology of alcohol teratogenesis, the current study focused on the quantification of CYP2E1 in prenatal human brain, a tissue that is highly vulnerable to the damaging effects of ethanol throughout gestation. In microsomal samples prepared from pools of brain tissues, immunoreactive protein was detected by Western blot analysis using enhanced chemiluminescence, whereas functional protein was estimated with an enzymatic assay using p-nitrophenol and an electrochemical detection system. CYP2E1 transcript was consistently detected in RNA samples prepared from individual brain tissues using the ribonuclease protection assay. Quantitative data were collected by scanning densitometry and phosphorimaging technology. There was a dramatic increase in human brain CYP2E1 content around gestational day 50 and a fairly constant level was maintained throughout the early fetal period, until at least day 113. The relatively low levels of the P-450 isoform present in conceptal brain may be sufficient to generate reactive intermediates that elicit neuroembryotoxicity following maternal alcohol consumption.

Inhibition of human prenatal biosynthesis of all-trans-retinoic acid by ethanol, ethanol metabolites, and products of lipid peroxidation reactions: a possible role for CYP2E1.

Biotransformation of all-trans-retinol (t-ROH) and all-trans-retinal (t-RAL) to all-trans-retinoic acid (t-RA) in human prenatal hepatic tissues (53-84 gestational days) was investigated with HPLC using human adult hepatic tissues as positive controls. Catalysis of the biotransformation of t-ROH by prenatal human cytosolic fractions resulted in accumulation of t-RAL with minimal t-RA. Oxidations of t-ROH catalyzed by prenatal cytosol were supported by both NAD+ and NADP+, although NAD+ was a much better cofactor. In contrast, catalysis of the oxidation of t-RAL to t-RA appeared to be solely NAD+ dependent. Substrate Km values for conversions of t-ROH to t-RAL and of t-RAL to t-RA were 82.4 and 65.8 microM, respectively. At concentrations of 10 and 90 mM, ethanol inhibited the conversion of t-ROH to t-RAL by 25 and 43%, respectively, but did not inhibit the conversion of t-RAL to t-RA significantly. In contrast, acetaldehyde reduced the conversion of t-RAL to t-RA by 25 and 87% at 0.1 and 10 mM respective concentrations. Several alcohols and aldehydes known to be generated from lipid peroxides also exhibited significant inhibition of t-RA biosynthesis in human prenatal hepatic tissues. Among the compounds tested, 4-hydroxy-2-nonenal (4-HNE) was highly effective in inhibiting the conversion of t-RAL to t-RA. A 20% inhibition was observed at a concentration of only 0.001 mM, and nearly complete inhibition was produced at 0.1 mM. Human fetal and embryonic hepatic tissues each exhibited significant CYP2E1 expression as assessed with chlorzoxazone 6-hydroxylation, a highly sensitive western blotting technique, and reverse transcriptase-polymerase chain reaction (PCR) (RT-PCR), suggesting that lipid peroxidation can be initiated via CYP2E1-catalyzed ethanol oxidation in human embryonic hepatic tissues. In summary, these studies suggest that ethanol may affect the biosynthesis of t-RA in human prenatal hepatic tissues directly and indirectly. Ethanol and its major oxidative metabolite, acetaldehyde, both inhibit the generation of t-RA. Concurrently, the CYP2E1-catalyzed oxidation of ethanol can initiate lipid peroxidation via generation of a variety of free radicals. The lipid peroxides thereby generated could then be further converted via CYP2E1-catalyzed reactions to alcohols and aldehydes, including 4-HNE, that act as potent inhibitors of t-RA synthesis.