Visible-Light-Activated Carbon Monoxide Release from Porphyrin-Flavonol Hybrids.

We report on porphyrin-flavonol hybrids consisting of a porphyrin antenna and four covalently bound 3-hydroxyflavone (flavonol) groups, which act as highly efficient photoactivatable carbon monoxide (CO)-releasing molecules (photoCORMs). These bichromophoric systems enable activation of the UV-absorbing flavonol chromophore by visible light up to 650 nm and offer precise spatial and temporal control of CO administration. The physicochemical properties of the porphyrin antenna system can also be tuned by inserting a metal cation. Our computational study revealed that the process occurs via endergonic triplet-triplet energy transfer from porphyrin to flavonol and may become feasible thanks to flavonol energy stabilization upon intramolecular proton transfer. This mechanism was also indirectly supported by steady-state and transient absorption spectroscopy techniques. Additionally, the porphyrin-flavonol hybrids were found to be biologically benign. With four flavonol CO donors attached to a single porphyrin chromophore, high CO release yields, excellent uncaging cross sections, low toxicity, and CO therapeutic properties, these photoCORMs offer exceptional potential for their further development and future biological and medical applications.

Structure-Photoreactivity Relationship of 3-Hydroxyflavone-Based CO-Releasing Molecules.

Carbon monoxide (CO) is an endogenous signaling molecule that regulates diverse physiological processes. The therapeutic potential of CO is hampered by its intrinsic toxicity, and its administration poses a significant challenge. Photoactivatable CO-releasing molecules (photoCORMs) are an excellent tool to overcome the side effects of untargeted CO administration and provide precise spatial and temporal control over its release. Here, we studied the CO release mechanism of a small library of derivatives based on 3-hydroxy-2-phenyl-4H-benzo[g]chromen-4-one (flavonol), previously developed as an efficient photoCORM, by steady-state and femto/nanosecond transient absorption spectroscopies. The main objectives of the work were to explore in detail how to enhance the efficiency of CO photorelease from flavonols, bathochromically shift their absorption bands, control their acid-base properties and solubilities in aqueous solutions, and minimize primary or secondary photochemical side-reactions, such as self-photooxygenation. The best photoCORM performance was achieved by combining substituents, which simultaneously bathochromically shift the chromophore absorption spectrum, enhance the formation of the productive triplet state, and suppress the singlet oxygen production by shortening flavonol triplet-state lifetimes. In addition, the cell toxicity of selected flavonol compounds was analyzed using in vitro hepatic HepG2 cells.

Photochemistry of (Z)-Isovinylneoxanthobilirubic Acid Methyl Ester, a Bilirubin Dipyrrinone Subunit: Femtosecond Transient Absorption and Stimulated Raman Emission Spectroscopy.

Bilirubin (BR) is an essential metabolite formed by the catabolism of heme. Phototherapy with blue-green light can be applied to reduce high concentrations of BR in blood and is used especially in the neonatal period. In this work, we studied the photochemistry of (Z)-isovinylneoxanthobilirubic acid methyl ester, a dipyrrinone subunit of BR, by steady-state absorption, femtosecond transient absorption, and stimulated Raman spectroscopies. Both the (Z)- and (E)-configurational isomers of isovinylneoxanthobilirubic acid undergo wavelength-dependent and reversible photoisomerization. The isomerization from the excited singlet state is ultrafast (the lifetimes of (Z)- and (E)-isomers were found to be ∼0.9 and 0.1 ps, respectively), and its efficiencies increase with increased photon energy. In addition, we studied sensitized photooxidation of the dipyrrinone subunit by singlet oxygen that leads to the formation of propentdyopents. Biological activities of these compounds, namely, effects on the superoxide production, lipoperoxidation, and tricarboxylic acid cycle metabolism, were also studied. Finally, different photochemical and biological properties of this BR subunit and its structural analogue, (Z)-vinylneoxanthobilirubic acid methyl ester, studied before, are discussed.

Common xanthene fluorescent dyes are visible-light activatable CO-releasing molecules.

Fluorescein, eosin Y, and rose bengal are dyes used in clinical medicine and considered (photo-)chemically stable. Upon extensive irradiation with visible light in aqueous solutions, we found that these compounds release carbon monoxide (CO) - a bioactive gasotransmitter - in 40-100% yields along with the production of low-mass secondary photoproducts, such as phthalic and formic acids, in a multistep degradation process. Such photochemistry should be considered in applications of these dyes, and they could also be utilized as visible-light activatable CO-releasing molecules (photoCORMs) with biological implications.

Antiproliferative and Cytotoxic Activities of Fluorescein-A Diagnostic Angiography Dye.

Fluorescein is a fluorescent dye used as a diagnostic tool in various fields of medicine. Although fluorescein itself possesses low toxicity, after photoactivation, it releases potentially toxic molecules, such as singlet oxygen (1O2) and, as we demonstrate in this work, also carbon monoxide (CO). As both of these molecules can affect physiological processes, the main aim of this study was to explore the potential biological impacts of fluorescein photochemistry. In our in vitro study in a human hepatoblastoma HepG2 cell line, we explored the possible effects on cell viability, cellular energy metabolism, and the cell cycle. We observed markedly lowered cell viability (≈30%, 75-2400 µM) upon irradiation of intracellular fluorescein and proved that this decrease in viability was dependent on the cellular oxygen concentration. We also detected a significantly decreased concentration of Krebs cycle metabolites (lactate and citrate < 30%; 2-hydroxyglutarate and 2-oxoglutarate < 10%) as well as cell cycle arrest (decrease in the G2 phase of 18%). These observations suggest that this photochemical reaction could have important biological consequences and may account for some adverse reactions observed in fluorescein-treated patients. Additionally, the biological activities of both 1O2 and CO might have considerable therapeutic potential, particularly in the treatment of cancer.

Cyanine-Flavonol Hybrids for Near-Infrared Light-Activated Delivery of Carbon Monoxide.

Carbon monoxide (CO) is an endogenous signaling molecule that controls a number of physiological processes. To circumvent the inherent toxicity of CO, light-activated CO-releasing molecules (photoCORMs) have emerged as an alternative for its administration. However, their wider application requires photoactivation using biologically benign visible and near-infrared (NIR) light. In this work, a strategy to access such photoCORMs by fusing two CO-releasing flavonol moieties with a NIR-absorbing cyanine dye is presented. These hybrids liberate two molecules of CO in high chemical yields upon activation with NIR light up to 820 nm and exhibit excellent uncaging cross-sections, which surpass the state-of-the-art by two orders of magnitude. Furthermore, the biocompatibility and applicability of the system in vitro and in vivo are demonstrated, and a mechanism of CO release is proposed. It is hoped that this strategy will stimulate the discovery of new classes of photoCORMs and accelerate the translation of CO-based phototherapy into practice.

Structural Modifications of Nile Red Carbon Monoxide Fluorescent Probe: Sensing Mechanism and Applications.

Carbon monoxide (CO) is a cell-signaling molecule (gasotransmitter) produced endogenously by oxidative catabolism of heme, and the understanding of its spatial and temporal sensing at the cellular level is still an open challenge. Synthesis, optical properties, and study of the sensing mechanism of Nile red Pd-based CO chemosensors, structurally modified by core and bridge substituents, in methanol and aqueous solutions are reported in this work. The sensing fluorescence "off-on" response of palladacycle-based sensors possessing low-background fluorescence arises from their reaction with CO to release the corresponding highly fluorescent Nile red derivatives in the final step. Our mechanistic study showed that electron-withdrawing and electron-donating core substituents affect the rate-determining step of the reaction. More importantly, the substituents were found to have a substantial effect on the Nile red sensor fluorescence quantum yields, hereby defining the sensing detection limit. The highest overall fluorescence and sensing rate enhancements were found for a 2-hydroxy palladacycle derivative, which was used in subsequent biological studies on mouse hepatoma cells as it easily crosses the cell membrane and qualitatively traces the localization of CO within the intracellular compartment with the linear quantitative response to increasing CO concentrations.

Wavelength-Dependent Photochemistry and Biological Relevance of a Bilirubin Dipyrrinone Subunit.

Phototherapy is a standard treatment for severe neonatal jaundice to remove toxic bilirubin from the blood. Here, the wavelength-dependent photochemistry of vinylneoxanthobilirubic acid methyl ester, a simplified model of a bilirubin dipyrrinone subunit responsible for a lumirubin-like structural rearrangement, was thoroughly investigated by liquid chromatography and mass and absorption spectroscopies, with the application of a multivariate curve resolution analysis method supplemented with quantum chemical calculations. Irradiation of the model chromophore leads to reversible Z → E photoisomerization followed by reversible photocyclization to a seven-membered ring system (formed as a mixture of diastereomers). Both the isomerization processes are efficient (ΦZE ∼ ΦEZ ∼ 0.16) when irradiated in the wavelength range of 360-410 nm, whereas the E-isomer cyclization (Φc = 0.006-0.008) and cycloreversion (Φ-c = 0.002-0.004) reactions are significantly less efficient. The quantum yields of all processes were found to depend strongly on the wavelength of irradiation, especially when lower energy photons were used. Upon irradiation in the tail of the absorption bands (490 nm), both the isomers exhibit more efficient photoisomerization (ΦZE ∼ ΦEZ ∼ 0.30) and cyclization (Φc = ∼0.07). In addition, the isomeric bilirubin dipyrrinone subunits were found to possess important antioxidant activities while being substantially less toxic than bilirubin.

Correction: The effect of light wavelength on in vitro bilirubin photodegradation and photoisomer production.

Following publication of this article, the authors noticed that an incorrect affiliation was assigned to the author "Lucie Muchová". The original article has now been updated so that the author "Lucie Muchová" is associated with the "Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Katerinská 32, 120 00 Prague, Czech Republic". This has been corrected in both the PDF and HTML versions of the article.

The effect of light wavelength on in vitro bilirubin photodegradation and photoisomer production.

BACKGROUND: The action spectrum for bilirubin photodegradation has been intensively studied. However, questions still remain regarding which light wavelength most efficiently photodegrades bilirubin. In this study, we determined the in vitro effects of different irradiation wavelength ranges on bilirubin photodegradation. METHODS: In our in vitro method, normalized absolute irradiance levels of 4.2 × 1015 photons/cm2/s from light-emitting diodes (ranging from 390-530 nm) and 10-nm band-pass filters were used to irradiate bilirubin solutions (25 mg/dL in 4% human serum albumin). Bilirubin and its major photoisomer concentrations were determined; the half-life time of bilirubin (t1/2) was calculated for each wavelength range, and the spectral characteristics for bilirubin photodegradation products were obtained for key wavelengths. RESULTS: The in vitro photodegradation of bilirubin at 37 °C decreased linearly as the wavelength was increased from 390 to 500 nm with t1/2 decreasing from 63 to 17 min, respectively. At 460 ± 10 nm, a significantly lower rate of photodegradation and thus higher t1/2 (31 min) than that at 500 nm (17 min) was demonstrated. CONCLUSION: In our system, the optimum bilirubin photodegradation and lumirubin production rates occurred between 490 and 500 nm. Spectra shapes were remarkably similar, suggesting that lumirubin production was the major process of bilirubin photodegradation.

Hyperbilirubinemia in Gunn Rats is Associated with Decreased Inflammatory Response in LPS-Mediated Systemic Inflammation.

Decreased inflammatory status has been reported in subjects with mild unconjugated hyperbilirubinemia. However, mechanisms of the anti-inflammatory actions of bilirubin (BR) are not fully understood. The aim of this study is to assess the role of BR in systemic inflammation using hyperbilirubinemic Gunn rats as well as their normobilirubinemic littermates and further in primary hepatocytes. The rats were treated with lipopolysaccharide (LPS, 6 mg/kg intraperitoneally) for 12 h, their blood and liver were collected for analyses of inflammatory and hepatic injury markers. Primary hepatocytes were treated with BR and TNF-α. LPS-treated Gunn rats had a significantly decreased inflammatory response, as evidenced by the anti-inflammatory profile of white blood cell subsets, and lower hepatic and systemic expressions of IL-6, TNF-α, IL-1ß, and IL-10. Hepatic mRNA expression of LPS-binding protein was upregulated in Gunn rats before and after LPS treatment. In addition, liver injury markers were lower in Gunn rats as compared to in LPS-treated controls. The exposure of primary hepatocytes to TNF-α with BR led to a milder decrease in phosphorylation of the NF-κB p65 subunit compared to in cells without BR. In conclusion, hyperbilirubinemia in Gunn rats is associated with an attenuated systemic inflammatory response and decreased liver damage upon exposure to LPS.

Valproic acid downregulates heme oxygenase-1 independently of Nrf2 by increasing ubiquitination and proteasomal degradation.

AIMS: Heme oxygenase-1 (HO-1; HMOX1 in human, Hmox1 in mice) is an antioxidative enzyme affecting wide range of sub-cellular processes. It was shown to modulate tumor growth or vascular-related diseases, thus being putative molecular target for tailored therapies. Therefore it is of importance to elucidate novel compounds regulating HO-1 activity/expression and to delineate mechanisms of their action. In the present study we aimed to understand mode of action of valproic acid (VA), an antiepileptic drug, on HO-1 expression. RESULTS: We demonstrated that HO-1 expression is decreased by VA at protein but not mRNA level in human alveolar rhabdomyosarcoma cell line CW9019. Nrf2 transcription factor, the activator of HO-1 expression through ARE sequence, was excluded as a mediator of HO-1 decrease, as VA downregulated Bach1, a Nrf2 repressor, concomitantly upregulating ARE activation. Also miRNA-dependent inhibition was excluded as a mechanism of HMOX1 regulation. However, co-immunoprecipitation assay showed a higher level of ubiquitinated HO-1 after VA treatment. Accordingly, MG132, an inhibitor of proteasomal degradation, reversed the effect of VA on HO-1 suggesting that decrease in HO-1 expression by VA is through protein stability. The inhibitory effect of VA on HO-1 was also observed in murine cells including embryonic fibroblasts isolated from Nrf2-deficient mice, what confirms Nrf2-independent effect of the compound. Importantly, VA decreased also HO-1 expression and activity in murine skeletal muscles in vivo. CONCLUSION: Our data indicate that VA downregulates HO-1 by acting through ubiquitin-proteasomal pathway leading to decrease in protein level.

Transition-Metal-Free CO-Releasing BODIPY Derivatives Activatable by Visible to NIR Light as Promising Bioactive Molecules.

Carbon monoxide-releasing molecules (CORMs) are chemical agents used to administer CO as an endogenous, biologically active molecule. A precise spatial and temporal control over the CO release is the major requirement for their applications. Here, we report the synthesis and properties of a new generation of transition-metal-free carbon monoxide-releasing molecules based on BODIPY chromophores (COR-BDPs) activatable by visible-to-NIR (up to 730 nm) light. We demonstrate their performance for both in vitro and in vivo experimental settings, and we propose the mechanism of the CO release based on steady-state and transient spectroscopy experiments and quantum chemical calculations.

Protective effect of heme oxygenase induction in ethinylestradiol-induced cholestasis.

Estrogen-induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase-1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol-induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 µmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down-regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up-regulated Mrp3 (348%, P ≤ 0.05). Heme pre-treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up-regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol-treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme-induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up-regulation (Mrp2/Mrp4) or down-regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2-dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol-induced cholestasis.

IL-1 receptor blockade alleviates endotoxin-mediated impairment of renal drug excretory functions in rats.

The aim of our study was to investigate whether two potent anti-inflammatory agents, dexamethasone and anakinra, an IL-1 receptor antagonist, may influence acute kidney injury (AKI) and associated drug excretory functions during endotoxemia (LPS) in rats. Ten hours after LPS administration, untreated endotoxemic rats developed typical symptoms of AKI, with reduced GFR, impaired tubular excretion of urea and sodium, and decreased urinary excretion of azithromycin, an anionic substrate for multidrug resistance-transporting proteins. Administration of both immunosuppressants attenuated the inflammatory response, liver damage, AKI, and increased renal clearance of azithromycin mainly by restoration of GFR, without significant influence on its tubular secretion. The lack of such an effect was related to the differential effect of both agents on the renal expression of individual drug transporters. Only dexamethasone increased the urinary clearance of bile acids, in accordance with the reduction of the apical transporter (Asbt) for their tubular reabsorption. In summary, our data demonstrated the potency of both agents used for the prevention of AKI, imposed by endotoxins, and for the restoration of renal drug elimination, mainly by the improvement of GFR. The influence of both drugs on altered tubular functions and the expression of drug transporters was differential, emphasizing the necessity of knowledge of transporting pathways for individual drugs applied during sepsis. The effect of anakinra suggests a significant contribution of IL-1 signaling to the pathogenesis of LPS-induced AKI.

Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds.

Spirulina platensis is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of S. platensis and S. platensis-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of S. platensis and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines in vitro in a dose-dependent manner (from 0.16 g•L-1 [S. platensis], 60 µM [PCB], and 125 µM [chlorophyllin], p<0.05). The anti-proliferative effects of S. platensis were also shown in vivo, where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (p < 0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (p = 0.0006; 0.016; and 0.006 for S. platensis, PCB, and chlorophyllin, respectively). In conclusion, S. platensis and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.

Cyanine-flavonol hybrids as NIR-light activatable carbon monoxide donors in methanol and aqueous solutions.

Here we report on carbon monoxide-photoreleasable compounds (photoCORMs) that combine heptamethine cyanine and flavonol chromophores and are activated upon irradiation with near-infrared light. Excellent CO-release yields and uncaging cross sections in aqueous solutions, enhanced water solubilities thanks to polar substituents or a host-guest approach using cucurbit[7]uril are demonstrated. The hybrids display outstanding biocompatibility and diverse, structure-dependent cell penetrability and internalization.

Bile acids decrease intracellular bilirubin levels in the cholestatic liver: implications for bile acid-mediated oxidative stress.

High plasma concentrations of bile acids (BA) and bilirubin are hallmarks of cholestasis. BA are implicated in the pathogenesis of cholestatic liver damage through mechanisms involving oxidative stress, whereas bilirubin is a strong antioxidant. We evaluated the roles of bilirubin and BA on mediating oxidative stress in rats following bile duct ligation (BDL). Adult female Wistar and Gunn rats intraperitoneally anaesthetized with ketamine and xylazine underwent BDL or sham operation. Cholestatic markers, antioxidant capacity, lipid peroxidation and heme oxygenase (HO) activity were determined in plasma and/or liver tissue 5 days after surgery. HepG2-rNtcp cells were used for in vitro experiments. Plasma bilirubin levels in control and BDL animals positively correlated with plasma antioxidant capacity. Peroxyl radical scavenging capacity was significantly higher in the plasma of BDL Wistar rats (210 ± 12%, P < 0.0001) compared to controls, but not in the liver tissues. Furthermore after BDL, lipid peroxidation in the livers increased (179 ± 37%, P < 0.01), whereas liver HO activity significantly decreased to 61% of control levels (P < 0.001). Addition of taurocholic acid (TCA, ≥ 50 µmol/l) to liver homogenates increased lipid peroxidation (P < 0.01) in Wistar, but not in Gunn rats or after the addition of bilirubin. In HepG2-rNtcp cells, TCA decreased both HO activity and intracellular bilirubin levels. We conclude that even though plasma bilirubin is a marker of cholestasis and hepatocyte dysfunction, it is also an endogenous antioxidant, which may counteract the pro-oxidative effects of BA in circulation. However, in an animal model of obstructive cholestasis, we found that BA compromise intracellular bilirubin levels making hepatocytes more susceptible to oxidative damage.

Role of heme oxygenase-1 in human endothelial cells: lesson from the promoter allelic variants.

OBJECTIVE: Heme oxygenase-1 (HO-1) is an antioxidative, antiinflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains a (GT)n microsatellite DNA, and the number of GT repeats can influence the occurrence of cardiovascular diseases. We elucidated the effect of this polymorphism on endothelial cells isolated from newborns of different genotypes. METHODS AND RESULTS: On the basis of HO-1 expression, we classified the HO-1 promoter alleles into 3 groups: short (S) (most active, GT < or = 23), medium (moderately active, GT=24 to 28), and long (least active, GT > or = 29). The presence of the S allele led to higher basal HO-1 expression and stronger induction in response to cobalt protoporphyrin, prostaglandin-J(2), hydrogen peroxide, and lipopolysaccharide. Cells carrying the S allele survived better under oxidative stress, a fact associated with the lower concentration of oxidized glutathione and more favorable oxidative status, as determined by measurement of the ratio of glutathione to oxidized glutathione. Moreover, they proliferated more efficiently in response to vascular endothelial growth factor A, although the vascular endothelial growth factor-induced migration and sprouting of capillaries were not influenced. Finally, the presence of the S allele was associated with lower production of some proinflammatory mediators, such as interleukin-1beta, interleukin-6, and soluble intercellular adhesion molecule-1. CONCLUSIONS: The (GT)n promoter polymorphism significantly modulates a cytoprotective, proangiogenic, and antiinflammatory function of HO-1 in human endothelium.

No association of promoter variations of HMOX1 and UGT1A1 genes with liver injury in chronic hepatitis C.

BACKGROUND: Heme oxygenase-1 (HMOX1) and bilirubin UDP-glucuronosyltransferase (UGT1A1), both enzymes involved in bilirubin homeostasis, play an important role in oxidative stress defense. OBJECTIVE: To assess the effect of promoter variations of HMOX1 and UGT1A1 genes on the progression of fibrosis in patients chronically infected with the hepatitis C virus (HCV). MATERIAL AND METHODS: The study was performed on146 chronic HCV infection patients, plus 146 age- and sex-matched healthy subjects. The (GT)n and (TA)n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis in all subjects. RESULTS: No differences were found in the frequencies of each particular allele of both genes, between HCV patients and a control group (p > 0.05). Furthermore, no association was detected (p > 0.05) between either the HMOX1 or the UGT1A1 promoter variants and the individual histological stages of liver disease in the HCV positive patients. Finally, no differences in the HMOX1 and UGT1A1 genotype prevalence rates were found between pre-cirrhotic and cirrhotic patients (p > 0.05). CONCLUSION: Based on our data, microsatellite variations in the HMOX1 and UGT1A1 genes are not likely to protect from progression of liver disease in patients with chronic hepatitis C.