Aldehyde dehydrogenase 2 polymorphism affects the outcome of methanol poisoning in exposed humans.

As the susceptibility of humans to xenobiotics often depends on genetic factors, we assumed that ADH1B and ALDH2 genetic variants may affect susceptibility to the acute methanol exposure. To evaluate the role of genetic variants of enzymes involved in methanol catabolism in humans, we analysed ADH1B (rs1229984) and ALDH2 (rs441) polymorphisms in 50 adults who survived acute methanol poisoning, 246 individuals with alcoholic liver cirrhosis, and in 545 healthy controls. GG homozygotes of ADH1B were more common among methanol-poisoned patients (98%) and among patients with alcoholic liver cirrhosis (98%) than among healthy controls (90%) (P = 0.08 and < 0.001, respectively). Minor C allele carriers of the ALDH2 were significantly more common among methanol-poisoned persons (46%) than among patients with alcoholic liver cirrhosis or healthy controls (31% in both groups, P < 0.05 and 0.025, respectively); the odds ratios were 1.89 (95% CI 1.02-3.52) and 1.94 (1.08-3.48), respectively. As there was a substantial amount of subjects with alcohol abuse between both groups of patients, ADH1B is unlikely to affect the susceptibility to methanol poisoning. By contrast, the genetic variant of the ALDH2 enzyme seems to specifically affect the susceptibility to methanol in acutely exposed humans and potentially plays a role in the outcome of methanol poisoning.

HNF-4α regulates expression of human ornithin carbamoyltransferase through interaction with two positive cis-acting regulatory elements located in the proximal promoter.

OTC encodes ornithine carbamoyltransferase, mitochondrial matrix enzyme involved in the synthesis of urea. The tissue-specific expression of OTC in the liver and intestine is dependent on the interaction of OTC promoter with an upstream enhancer. HNF-4 and C/EBPß are crucial for this interaction in the rat and mouse. In the present study we focused on characterization of elements involved in the regulation of OTC transcription in human. Using a set of 5'-deleted promoter mutants in a reporter assay we identified two positive cis-acting regulatory elements located at c.-105 and c.-136 within the human OTC promoter. Both are essential for the transcriptional activity of the promoter itself and for the interaction with the enhancer. Protein binding at the corresponding sites was confirmed by DNase I footprinting. Electromobility shift assay with a specific competitor and anti-HNF-4α antibody identified the DNA-protein binding sites as HNF-4α recognition motifs. A third HNF-4α binding site has been found at the position c.-187. All three HNF-4α binding sites are located within 35 bp upstream of the transcription start sites at positions c.-95, c.-119 (major) and c.-169 (minor). A series of C/EBPß recognition motifs was identified within the enhancer. Involvement of C/EBPß and HNF-4α in the promoter-enhancer interaction is further supported by a massive DNAprotein interaction observed in the footprinting and EMSA assays. Since the OTC promoter lacks general core promoter elements such as TATA-box or initiators in standard positions, HNF-4α most likely plays an essential role in the initiation of OTC transcription in human.

Ornithine carbamoyltransferase deficiency: molecular characterization of 29 families.

Ornithine carbamoyltransferase deficiency is the most common inherited defect of the urea cycle. We examined 28 male and 9 female patients from 29 families and identified 25 distinct mutations in OTC, 14 of which were novel. Three novel missense mutations (p.Ala102Pro, p.Pro158Ser, p.Lys210Glu) and a novel deletion of the Leu43 are not directly involved either in the enzyme active site or in the intersubunit interactions; however, the mutations include conserved residues involved in intramolecular interaction network essential for the function of the enzyme. Three novel large deletions - a 444 kb deletion affecting RPGR, OTC and TSPAN7, a 10 kb-deletion encompassing OTC exons 5 and 6 and a 24.5 kb-deletion encompassing OTC exons 9 and 10 - have probably been initiated by double strand breaks at recombination-promoting motifs with subsequent non-homologous end joining repair. Finally, we present a manifesting heterozygote carrying a hypomorphic mutation p.Arg129His in combination with unfavorably skewed X-inactivation in three peripheral tissues.

[Neonatal hyperbilirubinemia and molecular mechanisms of jaundice]. / Vrozené hyperbilirubinemie a molekulární mechanizmy zloutenky.

The introductory summarises the classical path of heme degradation and classification of jaundice. Subsequently, a description of neonatal types of jaundice is given, known as Crigler Najjar, Gilberts, DubinJohnson and Rotor syndromes, emphasising the explanation of the molecular mechanisms of these metabolic disorders. Special attention is given to a recently discovered molecular mechanism of the Rotor syndrome. The mechanism is based on the inability of the liver to retrospectively uptake the conjugated bilirubin fraction primarily excreted into the blood, not bile. A reduced ability of the liver to uptake the conjugated bilirubin contributes to the development of hyperbilirubinemia in common disorders of the liver and bile ducts and to the toxicity of xenobiotics and drugs using transport proteins for conjugated bilirubin.

[Evaluation of variants of unknown significance in the BRCA2 gene]. / Hodnocení variant nejasného významu v genu BRCA2.

BACKGROUND: Endogenous processes and exogenous agents cause constant DNA damage. DNA double-strand breaks are among the most serious types of damage. They are mainly repaired by homologous recombination, where the BRCA2 protein plays a dominant role. Heterozygous germline BRCA2 mutations predispose to breast, ovarian, pancreatic and other types of cancer. The presence of a pathogenic mutation in patients or their family members warrants close surveillance and prophylactic surgery. Apart from clearly pathogenic mutations, variants leading only to a single amino acid substitution are often identified. Since the influence of these variants on cancer risk is unknown, they represent a major clinical problem. AIMS: The aim of this paper is to summarize the current possibilities of predicting pathogenicity of BRCA2 variants. In some cases, genetic methods are able to classify variants with high probability; however, their use is often limited by low frequency of the variants or inaccessibility of samples for mRNA isolation or DNA from family members. Alternatively, functional assays performed in various cellular models may be employed. Multiple functional tests and cellular models are presented and characterized, including their advantages and limitations. A new model of human syngeneic cell lines developed by the authors is presented, in which one BRCA2 allele is deleted and the variant is introduced into the other allele by homologous recombination. This model has the potential to evaluate function of variants without some of the unwanted effects of the other models. Currently, this model is being applied to variants identified in patients with hereditary cancer predisposition in the Masaryk Memorial Cancer Institute. CONCLUSION: Functional assays in cellular models including a new model of syngeneic cell lines described by the authors have a great potential in evaluating clinical importance of unclassified variants in the BRCA2 gene, especially in cases where genetic tests are not applicable.

Portal hypertension is the main driver of liver stiffness in advanced liver cirrhosis.

Liver stiffness (LS) is a novel non-invasive parameter widely used in clinical hepatology. LS correlates with liver fibrosis stage in non-cirrhotic patients. In cirrhotic patients it also shows good correlation with Hepatic Venous Pressure Gradient (HVPG). Our aim was to assess the contribution of liver fibrosis and portal hypertension to LS in patients with advanced liver cirrhosis. Eighty-one liver transplant candidates with liver cirrhosis of various aetiologies underwent direct HVPG and LS measurement by 2D shear-wave elastography (Aixplorer Multiwave, Supersonic Imagine, France). Liver collagen content was assessed in the explanted liver as collagen proportionate area (CPA) and hydroxyproline content (HP). The studied cohort included predominantly patients with Child-Pugh class B and C (63/81, 77.8%), minority of patients were Child-Pugh A (18/81, 22.2%). LS showed the best correlation with HVPG (r=0.719, p< 0.001), correlation of LS with CPA (r=0.441, p< 0.001) and HP/Amino Acids (r=0.414, p< 0.001) was weaker. Both variables expressing liver collagen content showed good correlation with each other (r=0.574, p<0.001). Multiple linear regression identified the strongest association between LS and HVPG (p < 0.0001) and weaker association of LS with CPA (p = 0.01883). Stepwise modelling showed minimal increase in r2 after addition of CPA to HVPG (0.5073 vs. 0.5513). The derived formula expressing LS value formation is: LS = 2.48 + (1.29 x HVPG) + (0.26 x CPA). We conclude that LS is determined predominantly by HVPG in patients with advanced liver cirrhosis whereas contribution of liver collagen content is relatively low.

Regulation of diurnal variation of cholesterol 7alpha-hydroxylase (CYP7A1) activity in healthy subjects.

Cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory enzyme of bile acid synthesis, displays a pronounced diurnal variation. To better understand the regulation of CYP7A1 activity, three day-long examinations were carried out in 12 healthy men. The concentrations of 7alpha-hydroxycholest-4-en-3-one (C4), a surrogate marker of CYP7A1 activity, bile acids (BA), insulin, glucose, nonesterified fatty acids, triglycerides, and cholesterol were measured in serum in 90-min intervals from 7 AM till 10 PM. To lower and to increase BA concentration during the study, the subjects received cholestyramine and chenodeoxycholic acid (CDCA), respectively, in two examinations. No drug was used in the control examination. There was a pronounced diurnal variation of C4 concentration with a peak around 1 PM in most of the subjects. The area under the curve (AUC) of C4 concentration was five times higher and three times lower when subjects were treated with cholestyramine and CDCA, respectively. No relationship was found between AUC of C4 and AUC of BA concentration, but AUC of C4 correlated positively with that of insulin. Moreover, short-term treatment with cholestyramine resulted in about 10 % suppression of glycemia throughout the day. Our results suggest that insulin is involved in the regulation of diurnal variation of CYP7A1 activity in humans.

Role of common canalicular transporter gene variations in aetiology of idiopathic gallstones in childhood.

Variations in genes encoding canalicular transportes, for biliary lipids may affect concentrations of biliary lipids in bile and promote cholesterol crystallization and gallstone formation. In our study we investigated the contribution of heterozygosity for common variations considered either potentially pathogenic or susceptibility alleles for cholesterol cholelithiasis in adults (c.523A>G (p.Thr175Ala) and c.1954A>G (p.Arg652Gly) in ABCB4, c.1331T>C (p.Val444Ala) in ABCB11 and c.55 G>C (p.Asp19His) in ABCG8) to the aetiology of paediatric idiopathic gallstone disease. Genotyping was performed in 35 paediatric subjects with idiopathic gallstones with positive family history for gallstones and 150 population controls. The ABCB4 variant p.Thr175Ala was found only in the controls, not in the patients. The frequency of the remaining three variant alleles and the corresponding genotypes did not differ between patients and controls. We conclude that the studied common variations in genes encoding canalicular transporters known to contribute to genetic predisposition to cholesterol gallstones in adulthood do not contribute specifically to the aetiology of paediatric idiopathic gallstones.

Acute toxic effects of telmisartan in spontaneously hypertensive rats fed a high fructose diet.

Telmisartan is an angiotensin receptor blocker (ARB) and a selective peroxisome proliferator activated receptor gamma (PPARG) modulator. Recently, we tested metabolic effects of telmisartan (5 mg/kg body weight) in spontaneously hypertensive rats (SHR) fed a diet containing 60 % fructose, a widely used model of the metabolic syndrome. Surprisingly, we observed acute toxic effects of telmisartan. Rats lost body weight rapidly and died within 2 to 3 weeks due to bleeding into the upper gastrointestinal tract. SHR fed a high fructose diet and treated with telmisartan exhibited rapid decrease in blood pressure when compared to the SHR fed a high fructose diet and treated with valsartan. Concentrations of both unconjugated telmisartan and telmisartan glucuronide in the liver of SHR rats fed a high fructose diet were approximately 4 fold higher when compared to Brown Norway (BN) rats fed the same diet. Plasma concentrations of unconjugated telmisartan in the SHR were about 5 fold higher when compared to BN rats while plasma levels of telmisartan glucuronide were similar between the strains. Testing of other rat strains, diets, and the ARB valsartan showed that toxic effects of telmisartan in combination with high fructose diet are specific for the SHR. These results are consistent with the possibility that in some circumstances, SHR are predisposed to telmisartan toxicity possibly because of a genetically determined disturbance in telmisartan metabolism.

Large copy-number variations in patients with statin-associated myopathy affecting statin myopathy-related loci.

Some patients are susceptible to statin-associated myopathy (SAM) either because of genetic variations affecting statin uptake and metabolism, or because they predispose their carriers to muscular diseases. Among the frequent variants examined using the genome-wide association study approach, SLCO1B1 c.521T>C represents the only validated predictor of SAM in patients treated with high-dose simvastatin. Our aim was to ascertain the overall contribution of large copy-number variations (CNVs) to SAM diagnosed in 86 patients. CNVs were detected by whole genome genotyping using Illumina HumanOmni2.5 Exome BeadChips. Exome sequence data were used for validation of CNVs in SAM-related loci. In addition, we performed a specific search for CNVs in the SLCO1B region detected recently in Rotor syndrome subjects. Rare deletions possibly contributing to genetic predisposition to SAM were found in two patients: one removed EYS associated previously with SAM, the other was present in LARGE associated with congenital muscular dystrophy. Another two patients carried deletions in CYP2C19, which may predispose to clopidogrel-statin interactions. We found no common large CNVs potentially associated with SAM and no CNVs in the SLCO1B locus. Our findings suggest that large CNVs do not play a substantial role in the etiology of SAM.

[Fluorescence cystoscopy in the diagnostics and treatment of superficial urinary bladder tumors]. / Fluorescencní cystoskopie a její místo v diagnostice a lécbe povrchových nádoru mocového mechýre.

BACKGROUND: 5-aminolevulinic acid induced fluorescence cystoscopy can detect more tumour lesions comparing to standard cystoscopy. The goal of our study was to assess the influence of fluorescence cystoscopy used during transurethral resection on the recurrence rate and the length of tumor-free interval in stage Ta, Tl transitional cell carcinoma of the urinary bladder. METHODS AND RESULTS: In prospective randomized study 109 patients with primary or recurrent stage Ta Tl bladder transitional cell carcinoma treated with transurethral resection were enrolled. 17 patients with high grade tumors were evaluated separately. In group A the transurethral resection was performed with standard white light endoscopy, in group B with fluorescence cystoscopy. The patients were followed using standard cystoscopy and urinary cytology. Recurrence free interval was evaluated in whole groups and also for single and multiple and for primary and recurrent tumors separately. The median time to recurrence was 8.05 months in group A and was significantly shorter than 13.54 months in group B (p = 0.04, log-rank test). In separate analyses the median time to recurrence was significantly shorter using fluorescence cystoscopy in multiple (p = 0.004) and in recurrent (p = 0.02) tumors, but not in solitary and primary tumors. CONCLUSIONS: 5-aminolevulinic acid induced fluorescence cystoscopy used during transurethral resection reduces the early recurrence rate in stage Ta Tl bladder transitional cell carcinoma.

Role of biliary proteins and non-protein factors in kinetics of cholesterol crystallisation and gallstone growth.

Numerous biliary proteins as well as non-protein factors influence kinetics of cholesterol crystallisation from supersaturated bile. Cholesterol crystallisation pathways, methods used for quantitative evaluation of liquid-to-solid transition of cholesterol in bile and the classical concept of nucleation defect in pathogenesis of gallstones are reviewed in the first part of this article. The relevance of individual biliary proteins and non-protein factors is discussed in the second part. Finally, a new concept according to which accelerated crystallisation of cholesterol inhibits gallstone growth and protects the gallbladder wall from toxic injury resulting from the increased absorption and accumulation of cholesterol from supersaturated bile is suggested.

The effect of the irradiation wavelength on the processes sensitized by protoporphyrin IX dimethyl ester.

The formation of triplet states of photosensitizers and singlet oxygen during reactions sensitized by protoporphyrin IX dimethyl ester (PPDME) and the products of its photo-oxidation in solution were studied by time-resolved spectroscopy. Irradiation with long-wavelength light (670 nm), which is absorbed by the products of photo-oxidation of PPDME, provides lower quantum yields of singlet oxygen than in sensitization with PPDME alone, which absorbs light with a wavelength of 630 nm. Spectroscopic measurements also confirmed the lower rate of sensitized photo-oxidation of bilirubin during irradiation with light with a wavelength of 670 nm.

Influence of hyperbaric oxygenation on bilirubin and ditaurobilirubin auto-oxidation and porphyrin-sensitized photo-oxidation.

Oxygen concentration controls the reaction rate of porphyrin-sensitized photo-oxidation under constant experimental conditions. The rate of bilirubin and ditaurobilirubin auto-oxidation and meso-tetra-(4-sulphonatophenyl) porphine-sensitized photo-oxidation in aqueous solution increases by about 3.5 times in 0.5 MPa of oxygen. Use of hyperbaric oxygenation in the photodynamic therapy of tumours and in the phototherapy of jaundice is proposed.

Photodynamic therapy of cutaneous metastases of breast cancer after local application of meso-tetra-(para-sulphophenyl)-porphin (TPPS4).

Nine patients with cutaneous metastases of breast cancer were treated using photodynamic therapy. Meso-tetra-(para-sulphophenyl)-porphin (TPPS4) was applied locally at a dose of 0.15-0.3 mg directly to the lesion. The light source was an argon dye laser emitting light of 630 nm at a fluence rate of 312-680 mW cm-2 and a fluence of 150 J cm-2. No signs of local or systemic side effects or toxicity of the photosensitizer were observed. Treatments were well tolerated with no photosensitivity of the skin. Complete destruction of the tumour was observed in three patients, reduction of the tumour size by more than 50% in two patients, reduction-of the tumour size by less than 50% in two patients and no regression in two patients. The advantages of this method include the high concentration of the photosensitizer in the tumour, the extremely low total dose and the lack of side effects. The disadvantages include the less homogeneous distribution of the photosensitizer in the tumour tissue compared with that obtained by intravenous application.

Neurotoxicity of tetraphenylporphinesulfonate (TPPS4) and a hematoporphyrin derivative (Photosan) in organotypic cultures of chick embryonic dorsal root ganglia.

The neurotoxic effect of tetraphenylporphinesulfonate (TPPS4) and a hematoporphyrin derivative (HPD, Photosan) has been studied in organotypic cultures of chick dorsal root ganglia maintained in a semi-solid culture medium. The changes in two characteristics of neurite outgrowth, the mean radial length of neurites growing out from the ganglia and the area of neurite outgrowths, are used as parameters to evaluate the toxic effect. The porphyrins are tested over the concentration range 10-160 micrograms ml-1. TPPS4 is slightly more toxic than the HPD Photosan. The median inhibitory concentration (IC50) for TPPS4 is 45-50 micrograms ml-1 and for the HPD Photosan 50-60 micrograms ml-1, respectively. Nevertheless, the toxicity of the two drugs is relatively low compared to that of commonly used anticancer drugs, such as cisplatin or taxol.

Photodynamic effects of meso-tetra (4-sulfonatophenyl)porphine on human leukemia cells HEL and HL60, human lymphocytes and bone marrow progenitor cells.

Meso-tetra(4-sulfonatophenyl)porphine (TPPS4), in combination with a light dose of 14 J cm-2, has a profound negative effect on the proliferation and viability of leukemia cells HL60 (human promyelocytic leukemia) and HEL (human erythroleukemia), the viability of normal lymphocytes and the colony-forming activity of human bone marrow progenitor cells. However, normal leukocytes (monocytes, granulocytes) are, to a large extent, resistant to photodynamic treatment (PDT). Whilst DNA fragmentation suggesting apoptosis is induced in HL60 cells, accumulation in the interphase of the cell cycle (G0/G1, G2/M) is mainly operative in the TPPS4-mediated PDT of HEL cells. The "dark" effect of TPPS4 on the cell viability is below 15% up to a concentration of 40 microM.

Embryotoxicity of TPPS4 and PS 3 photosensitizers in chicken embryo under different light conditions.

Hematoporphyrin derivatives have been recommended for photodynamic therapy of malignant processes. We administered TPPS4, and Photosan 3 (PS 3) in chick embryo in ovo, with or without subsequent blue light (400-550 nm) irradiation. The aim was to analyze and compare the effects of both substances on organogenesis under different light conditions. The embryotoxic effect (embryonic death and malformations) was detected after a single intra-amniotic injection of 5 different doses (0.3 to 300 microg) of TPPS4 or PS 3 at embryonic day 3-5. The beginning of the embryotoxicity range (minimal embryotoxic dose) was determined in non-irradiated embryos to be between 0.3-3.0 microg PS 3 and 3.0-30.0 microg TPPS4. Malformations of surviving embryos were similar after both substances, represented by trunk hyperlordosis combined with incomplete closure of the ventral body wall and protrusion of viscera as consequences of amnion contraction, reduction limb deformities, eye malformations and cleft beak. Ten minutes light irradiation in ovo following two hours after intra-amniotic injection of TPPS4 or PS 3 increased by one order of magnitude their embryotoxic effects. Even dark-ineffective doses became highly embryotoxic. Contraction of the amniotic sac and extraembryonic vessels seemed to be a common mechanism of photosensitizer action.

Effect of chloroquine wash-out period of photosensitizers in the skin and selected organs in rats.

In the last decade, photodynamic therapy has become an alternative method for the diagnosis and therapy of tumors. In human medicine hematoporphyrin derivatives, sulfonated hydrophilic meso-tetraphenylporphyrin (TPPS4) and an oligomer of hematoporphyrin (Photosan 3), are widely used. Chloroquine is used for the treatment of porphyria cutanea tarda for its power to release porphyrins from the liver tissue. The kinetics of two porphyrin photosensitizers TPPS4 and Photosan 3 in the skin and some organs as well as the effect of chloroquine on the porphyrin excretion and their accumulation in skin and organs of Wistar rats were studied. TPPS4 exhibited maximum fluorescence in skin 48 h after application with decreasing to basal level from the 8th to the 14th day. Maximum fluorescence was reached at 72 hours after Photosan 3 application and it decreased to basal level during 96 hours after application. TPPS4 caused significantly higher fluorescence compared to Photosan 3. Chloroquine after oral administration did not change the fluorescence of skin, but it significantly decreased the TPPS4 concentration in rat organs if chloroquine treatment started 3 days or 2 weeks after TPPS4 application. Chloroquine significantly decreased the serum TPPS4 concentration during the period of 28 days. Fluorescence of skin was significantly higher and lasted longer after application of TPPS4 compared to Photosan 3. Chloroquine after oral administration did not influence the fluorescence of the skin, but it significantly decreased the TPPS4 concentration in rat organs. This effect could be useful in photodynamic therapy for mobilizing exogenous porphyrins from tissues after parenteral photodynamic therapy.