Extended neuroleptic administration modulates NMDA-R subunit immunoexpression in the rat neocortex and diencephalon.

BACKGROUND: This study aimed to evaluate the effect of extended olanzapine, clozapine and haloperidol administration on NMDA-R subunit immunoexpression in the rat neocortex and diencephalon. METHODS: To explore NR1, NR2A and NR2B subunit protein expression, densytometric analysis of immunohistochemically stained brain slices was performed. RESULTS: Interestingly, all neuroleptics caused a downregulation of NMDA-R subunit expression in the thalamus but increased the level of NR1 in the hypothalamus. Olanzapine upregulated hypothalamic NR2A expression, while clozapine and haloperidol decreased hypothalamic levels. We observed no significant changes in NR2B immunoreactivity. None of the studied medications had significant influence on NMDA-R subunit expression in the neocortex. CONCLUSIONS: Neuroleptic-induced reduction in the expression of thalamic NMDA-R subunits may play an important role in the regulation of glutamatergic transmission disorders in cortico-striato-thalamo-cortical loop in schizophrenia. A decrease in NMDA signaling in this region after long-term neuroleptic administration may also cautiously explain the incomplete effectiveness of these drugs in the therapy of schizophrenia-related cognitive disturbances.

Expression of cytochrome P450 2C and 3A in female rat liver after long-term administration of gonadoliberin analogs.

OBJECTIVES: Gonadoliberin (GnRH) analogs may be expected to indirectly modify growth hormone (GH) total concentration and its 24-h secretion profile. As a consequence, changes in the levels of GH may modify the mechanism of sex-dependent cytochromes P450 (CYP450) synthesis, including the expression of transcriptional factors. The aim of the study has been to evaluate the effect of long-term administration of a low dose of GnRH analogs on hepatic expression of CYP2C and CYP3A isoforms, and the transcription factors: pregnane X receptor (PXR), hepatocyte nuclear factor 4α (HNF4α), HNF6 and signal transducers and activators of transcription 5b (STAT5b). MATERIAL AND METHODS: The study was carried out on adult female Sprague-Dawley rats during a 3-month treatment with dalarelin (GnRH agonist) and cetrorelix (GnRH antagonist), at a daily intraperitoneal injection (i.p.) dose of 6 µg/kg body weight/day, and 1, 2, and 4 weeks after treatment discontinuation. The concentrations of ovarian hormones and GH in the blood serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) method, respectively. Then, the expression of hepatic CYP450s (reverse transcription polymerase chain reaction - RT-PCR, Western blot and immunohistochemistry) and transcription factors (RT-PCR) was evaluated. RESULTS: We have found that cetrorelix induces changes in the circadian pattern of GH secretion and enhances GH blood concentrations. These changes may cause increased expression of both, female-specific CYP450s (especially CYP3A9), and HNF4α/HNF6 transcription factors. Decrease in GH blood concentrations, resulting from the effect of dalarelin, may promote inhibition of female-specific CYP2C12 and CYP3A9 isoforms as well as STAT5b transcription factor. Slight changes in sex-independent CYP3A1 protein expression caused by GnRH analogs were also observed. CONCLUSIONS: In adult female rats, HNF4α/HNF6 and STAT5b seem to be crucial for the regulation of GnRH antagonist/GH- and GnRH agonist/GH-dependent pattern of CYP450 expression, respectively.

Developmental toxicity of hexachloronaphthalene in Wistar rats. A role of CYP1A1 expression.

Hexachloronaphthalene (HxCN) is one of the most toxic congeners of polychlorinated naphthalenes (PCNs). This study assesses the prenatal toxicity of HxCN after daily administration at doses of 0.1-1.0mg/kg b.w. to pregnant Wistar rats during organogenesis. We evaluated also the expression of CYP1A1 mRNA and protein in the livers of dams and fetuses, as well as the placenta. The results indicate that 0.3mg/kg b.w. was the lowest HxCN toxic dose for dams (LOAEL) while a dose of 0.1mg/kg b.w. was sufficient to impair the intrauterine development of embryos/fetuses without maternal toxicity. Regardless of the applied dose, HxCN generated embryotoxic effects. Dose-dependent fetotoxic effects were associated with HxCN exposure. HxCN was found to be a strong inducer of maternal and fetal CYP1A1. Expression of CYP1A1 mRNA in the placenta appears to be the most sensitive marker of HxCN exposure.

Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine.

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague-Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, ß-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b(5) reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.

Cytochrome P450 mRNA expressions along with in vitro differentiation of hepatocyte precursor cells from fetal, young and old rats.

Non-differentiated cells are attractive targets for cell therapy. During liver regeneration oval cells intensively proliferate and differentiate extending their metabolic activity. Hepatic cytochromes P450 (CYPs) can be linked either with metabolic activation of toxic compounds or drug metabolism. We investigated the differentiation and biotransformative potential of non-differentiated cells in primary cell cultures isolated from livers of fetuses (16-days-old), young (4-months-old) and old (20-months-old) rats. Under the conditions of experimental hepatocarcinogenesis, adult rats were fed for three weeks with CDE diet. Liver cells were cultured and precursor cells were differentiated to hepatocytes following induction with sodium butyrate (SB) or dimethyl sulphoxide (DMSO) in culture on MesenCult medium. We identified a number of cells expressing Thy-1, CD34, alpha-fetoprotein, cytokeratines--CK18 or CK19 and glutathione transferases--GSTpi or GSTalpha. In vitro differentiation of these cells, isolated from CDE-treated rats begun earlier as compared to non-treated ones. Age-dependent changes in the cell differentiation sequence, as well as CYPmRNA expression sequence accompanying precursor cells differentiation, were also observed. mRNA expression of CYP1A2, CYP2B1/2 and CYP3A1 was higher in the cells of young rats, but in the case of CYP2E1--in the cells of old rats. It was concluded that both proliferation and differentiation potential of oval cells, decreased with age.

Tobacco smoke-dependent changes in cytochrome P450 1A1, 1A2, and 2E1 protein expressions in fetuses, newborns, pregnant rats, and human placenta.

Tobacco smoke (TS) was described as a mixture of numerous cytochrome P450 (P450) substrates, inducers, and inhibitors. These inducers and inhibitors may modify drug clearance and xenobiotic or endogenous metabolism affecting P450s expression. In the present study, the effect of gestation and TS on: (1) cytochrome P450 CYP1A1, CYP1A2, and CYP2E1 protein expressions, and (2) cytochrome P450-linked microsomal enzyme activities, were studied in fetal rat liver, rat, and human placenta and in newborn and adult rat hepatic and extrahepatic tissues. Non-pregnant and pregnant 4-month-old female Wistar rats were exposed to TS (500, 1,000, or 1,500 mg carbon monoxide per m(3) air) in a toxicological chamber for 3 weeks (6 h daily, 5 days weekly). Human placentas were sampled from non-smoking, passive smoking, or active smoking primiparas. The efficacy of exposure was assessed by measuring urine cotinine levels. The TS-dependent inductory effect on the expression of CYP1A1 and 1A2 and related monooxygenase activities, and the inhibitory/inductory effect on CYP2E1 expression in rat tissues were observed. Pregnancy was associated with decreased levels of constitutive CYP1A1 and 2E1 in hepatic and extrahepatic tissues, TS-inducible CYP1A2 expression in the liver, and CYP1A1 expression in lungs and heart, but had no inhibitory effect on TS-inducible CYP1A1 and 2E1 expression, EROD, and P450-cooperated enzyme activities in the liver, kidney, and, in the latter case, in the heart. The presence of TS-induced CYP1A1 protein was confirmed in rat and human placenta and showed in newborn liver and lungs. CYP1A2 and 2E1 proteins were detectable in fetal rat liver. It was concluded that the expression of CYP1A1, 1A2, and 2E1, which metabolize some drugs and activate carcinogens, is controlled by age-, pregnancy-, and tissue-specific regulatory mechanisms in rats. Gestational differences in the regulation of expression of CYP1A subfamily members are not excluded. CYP1A1 and 2E1, but not CYP1A2 inductory mechanisms seem to be functional in fetal liver at day 21 of pregnancy but they appeared to be uninducible under a TS exposure. In TS-exposed pregnant females and fetuses the effects of metabolic activation of CYP1A1 and 1A2 substrates might be reduced because of lower CYP expressions or poor induction, respectively.