Piotr Czekaj

Relevance of glutathione S-transferase M1 and cytochrome P450 1A1 genetic polymorphisms to the development of head and neck cancers.

Abstract Background: Cytochrome P450 (CYP) and glutathione S-transferase (GST) gene variants have been intensively investigated for their implication in the development of different neoplasms. Methods: In the present study, we analyzed genetic polymorphisms of CYP1A1, GSTM1, GSTP1, and GSTT1 in 127 head and neck cancer patients and 151 hospital controls. Results: No significant increase in risk in patients with the GSTM1 null genotype (OR=1.52, 95% CI: 0.93–2.49) or CYP1A1 462Val alleles (OR=1.60, 95% CI: 0.73–3.52) or GSTP1 105Val alleles (OR=0.97, 95% CI: 0.59–1.58) was observed. The GSTT1 null genotype was found in 30.5% of the controls and 21.3% of the head and neck cancer patients (p=0.15). The estimated head and neck cancer risk for the combination of either CYP1A1 Ile462Val or CYP1A1 Val462Val genotype with either GSTP1 Ile105Val or Val105Val genotype (OR=2.89, 95% CI: 0.71–11.71) and for the combination of either CYP1A1 Ile462Val or CYP1A1 Val462Val genotype with GSTT1 null genotype (OR=2.62, 95% CI: 0.64–10.85) suggested the absence of the modifying effect of combined variant alleles on head and neck cancer susceptibility. The joint effect of either CYP1A1 Ile462Val or CYP1A1 Val462Val genotype with GSTM1 null genotype significantly increased the risk of head and neck cancer (OR=7.15, 95% CI: 1.49–34.32). Conclusions: Our findings corroborate metabolic genes interactions, especially for CYP1A1 462Val alleles and GSTM1 homozygous deletion, in the development of head and neck cancer in the investigated population groups in Poland. Clin Chem Lab Med 2008;46:1090–6.

Daily and circadian rhythms in the activity of mixed function oxidases system in rats of different age

Abstract An age‐associated alterations in daily and circadian changes of cytochrome P‐450‐linked monooxygenases system activity were studied using 1‐, 6‐ and 12‐months old male Wistar rats, in winter and in spring season. Cytochrome P‐450 and NADPH‐cytochrome P‐450 reductase showed 12h rhythm in all investigated age groups of animals. Cytochrome b5 and NADH‐cytochrome b5 reductase were characterized by a 12h daily rhythm in 1‐month old rats, but in older ones 24h circadian rhythm was found. There was not significant changes of the rhythm pattern in the activity of investigated MFO system ingredients in rats of different age, between spring and winter.

ERα36 – Another piece of the estrogen puzzle

Although the nuclear action of estrogen receptors (ER) is a well-known fact, evidence supporting membrane estrogen receptors is steadily accumulating. New ER variants of unrecognized function have been discovered. ERα is a product of the ESR1 gene. It serves not only as a template for the full-length 66kDa protein, but also for smaller isoforms which exist as independent receptors. The recently discovered ERα36 (36kDa), consisting of 310 amino acids of total 595 ERα66 protein residues, is an example of that group. The transcription initiation site is identified in the first intron of the ESR1 gene. C-Terminal 27 amino acids are encoded by previously unknown exon 9. The presence of this unique C-terminal sequence creates an opportunity for the production of selective antibodies. ERα36 has been shown to have a high affinity to the cell membrane and as much as 90% of the protein can be bound with it. Post-translational palmitoylation is suspected to play a crucial role in ERα36 anchoring to the cell membrane. In silico analysis suggests the existence of a potential transmembrane domain in ERα36. ERα36 was found in most cells of animals at various ages, but its exact physiological function remains to be fully elucidated. It seems that cells traditionally considered as being deprived of ER are able to respond to hormonal stimulation via the ERα36 receptor. Moreover, ERα36 displays unique pharmacological properties and its action may be behind antiestrogen resistance. The use of ERα36 in cancer diagnosis gives rise to great expectations.

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.

An experimental approach to the generation of human embryonic stem cells equivalents.

Recently, particular attention has been paid to the human embryonic stem cells (hESC) in the context of their potential application in regenerative medicine; however, ethical concerns prevent their clinical application. Induction of pluripotency in somatic cells seems to be a good alternative for hESC recruitment regarding its potential use in tissue regeneration, disease modeling, and drug screening. Since Yamanaka’s team in 2006 restored pluripotent state of somatic cells for the first time, a significant progress has been made in the area of induced pluripotent stem cells (iPSC) generation. Here, we review the current state of knowledge in the issue of techniques applied to establish iPSC. Somatic cell nuclear transfer, cell fusion, cell extracts reprogramming, and techniques of direct reprogramming are described. Retroviral and lentiviral transduction are depicted as ways of cell reprogramming with the use of integrating vectors. Contrary to them, adenoviruses, plasmids, single multiprotein expression vectors, and PiggyBac transposition systems are examples of non-integrative vectors used in iPSC generation protocols. Furthermore, reprogramming with the delivery of specific proteins, miRNA or small chemical compounds are presented. Finally, the changes occurring during the reprogramming process are described. It is concluded that subject to some limitations iPSC could become equivalents for hESC in regenerative medicine.

Morphological and enzymatic changes caused by a long-term treatment of female rats with a low dose of gonadoliberin agonist and antagonist

Summary Background Long-term treatment with gonadoliberin analogs is used to block the hypothalamic-pituitary-gonadal axis. The use of these agents is generally considered to be safe; however, some observations suggest the possibility of adverse effects. Material/Methods We investigated whether a 3-months administration of a low dose (6 μg/kg b.w.) of dalarelin – a new agonist, and cetrorelix – a known antagonist of GnRH to female rats causes morphological changes in pituitary gland, ovaries, uterus and liver (HE and VG staining); effects on pituitary, hepatic and blood enzyme activities (histochemical and kinetic methods, respectively), and on the blood lipid profile (colorimetric methods); and to what extent these changes are reversible. Results Applying analogs effectively inhibited ovulation, affected the uterine endometrium and changed histological appearance of the liver (e.g., steatosis). They altered activities of marker enzymes of cellular respiration, gluconeogenesis and intracellular digestion in the liver and, partially in the pituitary gland, caused undesirable changes in the activities of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and creatine kinase, and a concentration of cholesterol HDL fraction and triglycerides in the blood. Both morphological and enzymatic effects were more evident after antagonist administration; changes in the blood lipid profile were more evident after agonist administration. In both analogs histological and enzymatic changes persisted a relatively long time after the discontinuation of the treatment. Conclusions The low dose of dalarelin and cetrorelix is sufficient to cause limited damage of hepatic cells and may modify the function of pituitary, ovaries, uterus and liver as well as other organs, even after discontinuation of the treatment.

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.

Transcription Factors Potentially Involved in Regulation of Cytochrome P450 Gene Expression

Drug-metabolizing enzymes, including the cytochrome P450 (CYP) superfamily of enzymes, are subject to regulation by both exoand endogenous factors, mostly hormones and cytokines (Monostory et al., 2009; Waxman & Chang, 2005). In this regulation transcription factors are the mediators. Among them, orphan nuclear receptors: CAR (Constitutive Androstane Receptor), PXR (Pregnane X Receptor), VDR (Vitamin D Receptor), FXR (Farnesoid X Receptor), LXR (Liver X Receptor), PPAR┙ (Peroxisome Proliferator-Activated Receptor α) and RXR (Retinoid X Receptor) are the most important. They can create heterodimers in any configuration what, in conjunction with a broad spectrum of attached ligands, reflects the complexity of regulatory networks (Honkakoski & Negishi, 2000; Xu et al., 2005). Expression of some CYP isoforms is dependent on gender, which partly explains the metabolic difference between men and women in pharmacokinetics of drugs or, for instance, in susceptibility to carcinogens (Scandlyn et al., 2008). The main role in the sex-dependent regulation of CYP expression plays the growth hormone (GH) and to a lesser extent – other hormones. In principle, there are significant differences between genders in the daily profile of GH secretion into the bloodstream (Waxman & Chang, 2005). GH activates signaling pathway JAK-STAT (Lobie & Waxman, 2003). The main regulator of hepatic gene expression dependent on GH is transcription factor STAT5b which, together with other coregulators (i.e. HNF-4┙) can stimulate CYP genes directly by the binding to promoter sequences of target genes or indirectly by the activation of gene expressions of the genderspecific transcription factors (Park et al., 2006). As a result, in transactivation of cytochrome P450 genes, we can distinguish at least two pathways: (1) metabolic, dependent on the type of xenoor endobiotic, mediated by several nuclear receptors and (2) signaling, associated with activation of numerous GH-dependent transcription factors. Therefore, some endocrine disorders may cause changes in the drug metabolism, as well as in the CYP-dependent metabolism of endogenous substrates.

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.

What is the source of anticontractile factor released by the pedicle of human internal thoracic artery

OBJECTIVES Perivascular tissue (PVT) surrounding human internal thoracic artery (ITA) releases an unidentified anticontractile factor. The exact source of perivascular tissue-derived relaxing factor (PVRF) is unknown, although the adventitia and adipose tissue have both been suggested as primary candidates, hence the name adventitia or adipocyte-derived relaxing factor (ADRF). To look for the source of ADRF, we examined the dilatory response of human ITA to PVT aliquots in their histological composition. METHODS We studied isolated ITA segments from 20 patients subjected to coronary artery surgery. The vessels were skeletonized in vitro. ITA rings and PVT were incubated in separate isolated organ baths. The arterial rings were suspended on stainless steel wire hooks in the organ bath chamber. Vessel wall tension was measured with an isometric force transducer. Skeletonized ITA segments were precontracted with 10(-5.5) M phenylephrine. The 5 ml PVT aliquots were next transferred to the ITA tissue bath, resulting in its relaxation. Subsequently, the whole PVT used during experiment was fixed in paraformaldehyde and subjected to histological examination. Tissue was paraffin-embedded, sectioned and stained with haematoxylin and eosin. The paraffin blocks containing PVT were cut into slices every 800 μm to create three-dimensional model. Every PVT specimen was evaluated morphometrically using the Image Pro Plus software to assess the content of three basic kinds of tissues. The ITA relaxation to PVT aliquots was correlated to the histological composition of the PVT. RESULTS Phenylephrine elicited a 37.82 mN (Q1 = 26.49; Q3 = 46.31) contraction of the ITA. The addition of PVT aliquots to the skeletonized ITA induced a 54.17% (Q1 = 16.73; Q3 = 68.21) relaxation. The median PVT weight was 786 mg (Q1 = 562; Q3 = 976). The PVT composition was as follows: 30.5% (Q1 = 18.5; Q3 = 55.2) adipose tissue, 53.5 (Q1 = 24.6; Q3 = 66.5) muscular tissue and 13.5% (Q1 = 9.9; Q3 = 20.0) connective tissue. This translated into 197.7 mg of adipose tissue (Q1 = 142.2; Q3 = 393.2), 378.9 mg (Q1 = 178.8; Q3 = 537.0) of muscular tissue and 92.4 mg (Q1 = 68.6; Q3 = 185.8) of connective tissue. Neither PVT mass (r = 0.2, P = 0.92) nor adipose tissue (r = -0.2, P = 0.34), muscular tissue (r = 0.3, P = 0.18) or connective tissue (r = -0.2, P = 0.41) content correlated with ITA relaxation response to PVT aliquots. CONCLUSIONS Adipose tissue from the pedicled ITA graft is an unlikely source of ADRF.