Imipramine demethylation in vivo: impact of CYP1A2, CYP2C19, and CYP3A4

Nephron loss leads to increased production of reactive oxygen intermediates. We measured the effect of carvedilol, a beta-blocking drug with radical scavenging properties, on renal function, glomerulosclerosis, antioxidant enzyme status and in vivo hydrogen peroxide (H2O2) production in rats with chronic renal failure caused by 5/6 nephrectomy (remnant kidney) and compared results to data obtained with propranolol, a beta-blocking drug without scavenging characteristics. Carvedilol and propranolol were administered during 11 weeks following reduction of nephron number. Kidneys were examined using enzymatic and histological techniques. Both carvedilol and propranolol decreased systolic blood pressure. Compared to propranolol, carvedilol offered some additional beneficial effects on renal function, particularly with regard to glomerulosclerosis. Lipid peroxidation, evaluated by malonaldehyde and 4-hydroxynonenal concentration in cortex homogenates, was decreased in carvedilol-treated rats only. Superior beneficial effect of carvedilol treatment is not linked to a significant up-regulation of the activities of the remnant kidney antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase) or to a decreased in vivo H2O2 production.     

Differential in vivo effects of alpha-naphthoflavone and beta-naphthoflavone on CYP1A1 and CYP2E1 in rat liver, lung, heart, and kidney

Tenascin is a large extracellular matrix glycoprotein expressed in neural and non-neural tissues. In the central nervous system, tenascin is synthesized by astrocytes during development and wound healing, forming barriers and affecting neurite outgrowth. In this study we examined tenascin expression in optic nerve heads of normal and glaucomatous eyes and found that there is upregulation of tenascin mRNA and protein in reactive astrocytes from human glaucomatous optic nerve heads compared to normal age-matched controls. In the prelaminar region there was a band of tenascin immunoreactivity around the blood vessels of glaucomatous, but not in normal eyes. However, tenascin mRNA was only localized to astrocytes, suggesting that astrocytes are the cellular source of tenascin. In the lamina cribrosa, tenascin immunoreactivity and gene expression were localized to astrocytes in the cribriform plates and inside the nerve bundles. In the post-lamina region, tenascin immunoreactivity and gene expression were localized to astrocytes lining the pial septum immediately adjacent to the lamina cribrosa. In normal optic nerve heads, tenascin expression at the mRNA and protein levels was confined to clusters of astrocytes at the level of Bruch's membrane in the prelaminar optic nerve head. In glaucoma, enhanced expression of tenascin may be protective to the axons of the retinal ganglion cells by providing a barrier for humoral and/or blood-borne factors that may cause further neural damage. However, the precise role of tenascin in glaucomatous optic neuropathy is not yet elucidated.     

Inhibition of cytochrome P-450 3A (CYP3A) in human intestinal and liver microsomes: comparison of Ki values and impact of CYP3A5 expression

Seckel syndrome has been described as the prototype of the primordial bird-headed type of dwarfism. Since Seckel originally defined the disorder, less than 60 cases have been reported. In addition to the characteristic craniofacial dysmorphism and skeletal defects, abnormalities have been described in the cardiovascular, hematopoietic, endocrine, and central nervous systems. This pleiotropy has implied genetic heterogeneity and prompted reviews of previously reported cases of Seckel syndrome. As a result, the characteristic diagnostic features of Seckel syndrome have been highly debated. Although deletions in chromosome 2q have been described, to date, no genetic defect has been defined. We report three cases of Seckel-like syndrome in siblings from nonconsanguinous Caucasian parents. In addition to the typical Seckel phenotypic features, all three cases were characterized by severe hydrocephalus. We review the literature and propose that there is a spectrum of Seckel conditions that share some common key features, but also demonstrate a wide range of phenotypic features.     

Detection and localization of CYP1A1/CYP1A2 in murine liver: electrophoresis, immunoblotting and immunocytochemistry

Multiple forms of cytochrome P450 exist some of which are selectively inducible by exposure of the organism to a variety of foreign compounds. In this study, a monoclonal antibody specific for 3-methyl-cholanthrene-inducible cytochrome P450, Mab 1-7-1, was used to detect, localize and quantify CYP1A1/CYP1A2 in livers of C57BL/6 mice. Mab 1-7-1 recognized a faint band in the range between 45-66 Kd in Western immunoblots of liver microsomes from control mice, and a strong band in the same range, in liver microsomes from beta-naphthoflavone-treated mice. Microsome from control liver contained minimal levels of CYP1A1/CYP1A2; pretreatment with beta-naphthoflavone caused an increase in their expression. Immunoelectron microscopy was used to demonstrate the cellular localization and quantification of these isozymes in the liver. The immunolabeling procedure confirmed the endoplasmic reticulum as the primary site of CYP1A1/CYP1A2 induction in hepatocytes. This organelle showed the highest labeling density after treatment with beta-naphthoflavone. Increase in CYP1A1/CYP1A2 was 33.4-fold by morphometric analysis in induced hepatocytes in comparison to non-induced cells. In conclusion, CYP1A1/CYP1A2 is highly induced by beta-naphthoflavone in C57BL/6 mouse liver, and the cellular site of expression is the endoplasmic reticulum.     

Stimulation of 5-HT1A receptor inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo

Phosphatidylinositol synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyl-transferase, EC 2.7.8.11) is a 24-kDa membrane-bound enzyme. It is present in all mammalian cells and is localized predominantly to the endoplasmic reticulum. The enzyme performs the last step in the de novo biosynthesis of the phospholipid phosphatidylinositol by catalyzing the condensation of CDP-diacylglycerol and myo-inositol to form the products phosphatidylinositol and CMP. Phosphatidylinositol, apart from being an essential membrane phospholipid, is involved in protein membrane anchoring and is the precursor for the second messengers inositol-tri-phosphate and diacylglycerol.     

Evidence for the presence of adrenergic receptors in 3-day-old chick embryo

Effects of sympathomimetic amines without and with alpha and beta-adrenergic blocking agents on the heart rate and arterial and venous blood pressures in the 3-day-old chick embryo were studied. No chronotropic effect was observed. Norepinephrine caused a biphasic change in systolic and diastolic arterial pressures, the lower doses effecting a fall, and the higher doses a rise in these pressures. With phenylephrine a sharp rise in systolic, diastolic, and pulse pressures was seen. Isoproterenol caused a dramatic fall in systolic, diastolic, and pulse pressures. In the presence of phenoxybenzamine, the pressor effect of high doses of norepinephrine was reversed, the pressor effect of phenylephrine was abolished, and the hypotension with isoproterenol was enhanced. After propranolol, the hypotensive effect of low doses of norepinephrine was reversed, the pressor response to phenylephrine was unchanged, and the depressor effect of isoproterenol was abolished. These findings suggest the presence of functioning alpha- and beta-receptors in the 3-day-old chick embryo. Additionally, they suggest that the alpha-receptors develop more slowly in the chick embryo.     

Developmental expression of CYP2C and CYP2C-dependent activities in the human liver: in-vivo/in-vitro correlation and inducibility

Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed. We have localized the epitope of the anti-porcine VCAM blocking mAbs 2A2 and 3F4 to domains 1 and 2, respectively. Humanized antibodies (IgG4 isotype) were constructed from these anti-porcine VCAM antibodies and demonstrated to inhibit adhesion of Ramos, Jurkat and YT cells, as well as purified resting and activated human T cells, to porcine aortic endothelial cells (PAEC). These cell types express both LFA-1 as well as VLA-4, suggesting blockade of human VLA-4 interaction with porcine VCAM may alone be sufficient to significantly impair adhesion of human leukocytes to porcine endothelial cells. The chimeric anti-porcine VCAM (pVCAM) HuG4 antibodies promoted increased adhesion of Fc receptor (FcR) positive cells such as U937 monocytic cells to PAEC. In contrast, chimeric anti-porcine VCAM antibodies created using the CH1 and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4 (HuG2/G4 antibodies) inhibited binding of FcR positive cells to PAEC. These chimeric anti-pVCAM antibodies should allow delineation of the in vivo role of VLA-4/VCAM interaction in porcine-to-primate xenotransplants. Further, the design of the HuG2/G4 antibodies should render them efficacious in multiple settings requiring elimination of FcR binding.     

Different embryotoxic effect of vitamin A and B-carotene detected in the chick embryo

Teratogenicity of vitamin A was firstly detected in experimental animals in 1953. Nearly 30 years later, teratogenicity of vitamin A analogue-isotretinoin was reported in humans. Isotretinoin induces serious birth defects of craniofacial and central nervous system, cardiovascular system and thymic malformations--in about 25% of babies exposed during the first trimester of their prenatal development. The biological interconversion of isotretinoin to vitamin A is known. That is why later epidemiological studies focused on high vitamin A intake in pregnant woman: Women who use daily vitamin A supplements during early pregnancy have approximately a two-fold increased risk of giving birth to a malformed baby. On the basis of these data, replacement of vitamin A has been recommended with its natural precursor B-carotene which is supposed to be more safe for pregnant woman due to its limited absorption from intestine. Aim of the present paper was to test a possible direct embryotoxic effect (i.e. lethality + teratogenicity) of B-carotene in chick embryos and to compare these results with known embryotoxicity of vitamin A in the same experimental model. Single subgerminal or intaamniotic injection of vitamin A or B-carotene within day 2-5 of incubation was used for estimation of the beginning of the embryotoxicity range determining the minimal embryotoxic doses. Vitamin A started to affect development between doses 0.3-0.3 microm [corrected] per embryo. Malformations of head, extremities and heart were detected similarly like in laboratory mammals and in man. B-carotene exhibited such an effect neither after injection of the highest tested doses-100 microm [corrected] per embryo. The results documented the strong difference in embryotoxicity between vitamin A and B-carotene. After theoretical extrapolation of the results achieved in the chick embryo, the minimal embryotoxic doses of vitamin A in mammals were estimated to be between 0.1-1 mg/kg of maternal weight and those of B-carotene to be more than 1000 mg/kg of maternal weight. Human epidemiological studies have proved teratogenicity of vitamin A after daily doses 25,000 i.u.-8.3 mg (0.13 mg/kg)- and reduction of its maximum intake has been recommended to 10,000 i.u. per day (0.05 mg/kg). The results about teratogenicity of vitamin A achieved in the chick embryo are in agreement with such a recommendation. Intake of vitamin A in the food is sufficient for pregnant woman in common Czech population. Therefore, an artificial supplementation of vitamin A brings risk of overdosage. If supplementation by vitamin A is unavoidable during pregnancy, B-carotene should be preferred.     

Validation of precision-cut liver slices in dynamic organ culture as an in vitro model for studying CYP1A1 and CYP1A2 induction

The utilization of precision-cut liver slices in dynamic organ culture as an in vitro model was validated by comparing the induction of the biomarker responses following in vitro (rat liver slice) and in vivo exposure of rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The biomarker responses investigated were cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) mRNA, protein, and activities. Precision-cut rat liver slices were incubated in dynamic organ culture for 24 hr with medium containing 0.001-10 nM TCDD or medium without TCDD (control). The resultant mean TCDD concentration in the slices ranged from 19 to 80,925 ppt (wet wt), respectively. A concentration-dependent induction of CYP1A1 mRNA, protein, and activities and a more modest induction of CYP1A2 mRNA was observed in liver slices at all medium concentrations of TCDD. The O-demethylation of 7-methoxyresorufin, a marker for CYP1A2 activity, was induced at TCDD medium levels of 0.01 nM and greater, whereas a detectable increase in CYP1A2 protein occurred only at the higher concentrations. Comparable liver concentrations of TCDD (8-64,698 ppt wet wt) were achieved at 24 hr following a single in vivo exposure of rats to TCDD at doses ranging from 0.002 to 5 microg/kg po. Concentration-effect and dose-response relationships for induction of CYP1A1 and CYP1A2 were similar following in vitro and in vivo exposure to TCDD, although the magnitude of induction was greater for in vivo exposure. The data support the use of liver slices in dynamic organ culture for assessing the relative in vivo potency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in this in vitro model to predict the biological and toxicological effects of a given in vivo exposure to TCDD.     

Differential regulation of gastrulation and neuroectodermal gene expression by Snail in the Drosophila embryo

The initiation of mesoderm differentiation in the Drosophila embryo requires the gene products of twist and snail. In either mutant, the ventral cell invagination during gastrulation is blocked and no mesoderm-derived tissue is formed. One of the functions of Snail is to repress neuroectodermal genes and restrict their expressions to the lateral regions. The derepression of the neuroectodermal genes into the ventral region in snail mutant is a possible cause of defects in gastrulation and in mesoderm differentiation. To investigate such possibility, we analysed a series of snail mutant alleles. We found that different neuroectodermal genes respond differently in various snail mutant background. Due to the differential response of target genes, one of the mutant alleles, V2, that has reduced Snail function showed an intermediate phenotype. In V2 embryos, neuroectodermal genes, such as single-minded and rhomboid, are derepressed while ventral invagination proceeds normally. However, the differentiation of these invaginated cells into mesodermal lineage is disrupted. The results suggest that the establishment of mesodermal cell fate requires the proper restriction of neuroectodermal genes, while the ventral cell movement is independent of the expression patterns of these genes. Together with the data showing that the expression of some ventral genes disappear in snail mutants, we propose that Snail may repress or activate another set of target genes that are required specifically for gastrulation.     

A method for recording the respiratory and hatching movements of the chick embryo

1. An artificial air space was made in the narrow end of the pipped egg by separating the shell membranes from the shell. The respiratory and hatching movements of the embryo set up pressure changes within the artificial air space and these were recorded up until the moment when the chick emerged from the shell. 3. Random movements appeared to be inhibited during the period of hatching. 4. The ways in which the presence of the artificial air space might affect the embryo have been discussed.     

Apolipoprotein (apo) B and apoII gene expression are both estrogen-responsive in chick embryo liver but only apoII is estrogen-responsive in kidney

Estrogen regulates the hepatic synthesis of a variety of proteins required for egg yolk production in oviparous vertebrates. In chickens, two of these proteins, apolipoprotein (apo) B and apoII, comprise the major protein components of specialized very low density lipoprotein particles that transport triacylglycerols and cholesterol to the developing egg yolk. In the adult, apoB is synthesized constitutively in liver, small intestine, and kidney but is estrogen-responsive only in the liver. In this work we have examined the embryonic expression of the apoB and apoII genes in yolk sac, liver, kidney, and small intestine. The 14 kb apoB mRNA was first detected at day 3 of development in vascular yolk sac, a tissue involved in the transfer of yolk lipids into the embryonic circulation. Constitutive apoB mRNA expression was detectable in liver at day 6.5 and in kidney at day 7.5, but in intestine was barely detectable before hatching. The hepatic apoB gene acquired estrogen-responsiveness at day 6.5 and its hormone-dependent expression increased throughout development in concert with the estrogen-responsive expression of the apoII gene. In contrast, the constitutively expressed apoB gene in kidney remained unresponsive to estrogen. Surprisingly, the apoII gene was found to be responsive to estrogen in both the embryonic kidney and small intestine. ApoII mRNA induction by estrogen in kidney at day 11 was at 10% of the level in the liver but estrogen-responsiveness decreased later in development and was low in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of administration of the chemoprotective agent oltipraz on CYP1A and CYP2B in rat liver and rat hepatocytes in culture

The success of oltipraz (OPZ) [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] as a chemoprotective agent against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat is thought to depend principally on its ability to enhance detoxication by inducing phase II enzymes, especially glutathione transferases. However, in primary cultures of human hepatocytes, we recently demonstrated that OPZ also has an important inhibitory effect on the major cytochromes P450 (CYPs) of human hepatic AFB1 metabolism. This has prompted a detailed study of the effect of OPZ on some CYPs involved in metabolism of AFB1 in the rat. Primary cultures of rat hepatocytes behaved similarly to human hepatocytes and responded to OPZ by inhibition of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD) activities mainly associated, respectively, with CYP1A and CYP2B. A time-course shows that this inhibition is largely reversible, with EROD and PROD activities reaching a minimum at 12 h and tending towards control values within 24 h. As is to be expected, the incubation of isolated microsomes with OPZ also inhibits CYP1A and 2B. The effect of OPZ on CYP1A is not a phenomenon limited to cells in culture, but also occurs in vivo. Using the whole animal, we were able to demonstrate that OPZ also transiently inhibited CYP1A activity in a rat given caffeine, by measuring the amounts of methylxanthines found in the serum. However, microsomes isolated from rats, that had been treated with OPZ in vivo, show no such inhibition, presumably because, since OPZ is a reversible inhibitor, it dissociates and is lost during the course of conventional procedures of microsomal preparation. This explains some earlier failures in studies of isolated microsomes to observe the inhibition of CYPs by OPZ. In addition to inhibiting their enzymatic activity, OPZ is also an inducer of CYP1A and 2B as shown by the increased levels of their mRNAs and of caffeine metabolism in vivo after 24 h or more. It is concluded that the mechanism of chemoprotection by OPZ, of toxic chemical metabolism in the rat, is complex and involves competitive inhibition of activation succeeded by induction of the enzymes of both activation and detoxication.