Lenka Skálová

Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep.

In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins cross-reacting with anti-human CYP3A4, CYP2C9, and CYP2E1 IgG was detected in all farm species. Bovine microsomes differed from other microsomes of farm species in very high 7-ethoxyresorufin-O-deethylase activity (CYP1A1/2). Significantly higher 7-methoxy-4-trifluoromethyl-coumarin demethylase (2-3 times) and 12-lauric acid hydroxylases (4-10 times) activities (probably corresponding to CYP2C and CYP4A, respectively) were found in ovine microsomes. The highest 6beta-testosterone hydroxylase activity, which is usually considered to be a CYP3A activity marker, was found in pig. Reductases of all farm animals display considerable ability to reduce carbonyl group of xenobiotics. Significant differences in level and activity of many biotransformation enzymes tested suggest that extrapolation of pharmacokinetic data obtained in one species to another (even related) could be misleading.

Chiral Inversion of Drugs: Coincidence or Principle?

2-arylpropionic acid derivatives are probably the most frequently cited drugs exhibiting the phenomenon that is best known as chiral inversion. One enantiomer of drug is converted into its antipode either in the presence of a solvent or more often in inner environment of an organism. Mechanistic studies of the metabolic chiral inversion were carried out for several drugs from NSAIDs, and a model of this inversion was suggested and subsequently confirmed. The chiral inversion of NSAIDs has been intensively studied in the context of the pharmacological and toxicological consequences. However, the group of NSAIDs is not the sole group of drugs in which the inversion phenomenon can be observed. There exist several other drugs that also display chiral inversion of one or even both of their enantiomers. These drugs belong to different pharmacotherapeutic groups as monoamine oxidase inhibitors, antiepileptic drugs, drugs used in the treatment of hyperlipoproteinemia or drugs that are effective in the treatment of leprosy. Moreover, some chiral or prochiral drugs are metabolized to give chiral metabolites that undergo chiral inversion too, which can have direct impact on pharmacological properties or toxicity of the drug. As the process of chiral inversion is affected by several factors, so the intensity of chiral inversion of individual substances and at different conditions can differ considerably. Interspecies differences and types of tissue are reported to be the main factors that were recognized to play the key role in the process of chiral inversion. Some of more recent studies have revealed that several other factors, such as the route of administration or interaction with other xenobiotics, can influence the enantiomeric conversion, too. Chiral inversion does not seem to be a phenomenon connected with only several drugs from some unique group of 2-arylpropionic acid derivatives: it is also observed in drugs with rather different chemical structures and is much more frequent than it can be realized.

Veterinary drugs in the environment and their toxicity to plants.

Veterinary drugs used for treatment and prevention of diseases in animals represent important source of environmental pollution due to intensive agri- and aquaculture production. The drugs can reach environment through the treatment processes, inappropriate disposal of used containers, unused medicine or livestock feed, and manufacturing processes. Wide scale of veterinary pharmaceuticals e.g. antibiotics, antiparasitic and antifungal drugs, hormones, anti-inflammatory drugs, anaesthetics, sedatives etc. enter the environment and may affect non-target organisms including plants. This review characterizes the commonly used drugs in veterinary practice, outlines their behaviour in the environment and summarizes available information about their toxic effect on plants. Significant influence of many antibiotics and hormones on plant developmental and physiological processes have been proved. However, potential phytotoxicity of other veterinary drugs has been studied rarely, although knowledge of phytotoxicity of veterinary drugs may help predict their influence on biodiversity and improve phytoremediation strategies. Moreover, additional topics such as long term effect of low doses of drugs and their metabolites, behaviour of mixture of veterinary drugs and other chemicals in ecosystems should be more thoroughly investigated to obtain complex information on the impact of veterinary drugs in the environment.

Xenobiotic metabolizing enzymes and metabolism of anthelminthics in helminths.

Anthelminthics remain the only accessible means in the struggle against helminth parasites, which cause significant morbidity and mortality in man and farm animals. The treatment of helminthic infections has become problematic because of frequent drug resistance of helminth parasites. The development of drug resistance can be facilitated by the action of xenobiotic metabolizing enzymes (XMEs). In all organisms, XMEs serve as an efficient defense against the potential negative action of xenobiotics. The activities of XMEs determine both desired and undesired effects of drugs, and the knowledge of drug metabolism is necessary for safe, effective pharmacotherapy. While human and mammalian XMEs have been intensively studied for many years, XMEs of helminth parasites have undergone relatively little investigation, so far. However, many types of XMEs, including oxidases, reductases, hydrolases, transferases, and transporters, have been described in several helminth species. XMEs of helminth parasites may protect these organisms from the toxic effects of anthelminthics. In case of certain anthelminthics, metabolic deactivation was reported in helminth larvae and/or adults. Moreover, if a helminth is in the repeated contact with an anthelminthic, it defends itself against the chemical stress by the induction of biotransformation enzymes or transporters. This induction can represent an advantageous defense strategy of the parasites and may facilitate the drug-resistance development.

Reduction of doxorubicin and oracin and induction of carbonyl reductase in human breast carcinoma MCF-7 cells.

In cancer cells, the drug-metabolizing enzymes may deactivate cytostatics, thus contributing to their survival. Moreover, the induction of these enzymes may also contribute to development of drug-resistance through acceleration of cytostatics deactivation. However, the principal metabolic pathways contributing to deactivation of many cytostatics still remain poorly defined. The main aims of the present study were: (i) to compare the reductive deactivation of cytostatic drugs doxorubicin (DOX) and oracin (ORC) in human breast cancer MCF-7 cells; (ii) to identify major enzyme(s) involved in the carbonyl reduction; and iii) to evaluate the activities and expression of selected carbonyl reducing enzymes in MCF-7 cells upon a short-term (48 h) exposure to either DOX or ORC. We found that MCF-7 cells were able to effectively metabolize both DOX and ORC through reduction of their carbonyl groups. The reduction of ORC was stereospecific, with a preferential formation of + enantiomer of dihydrooracin (DHO). The cytosolic carbonyl reductase CBR1 seemed to be a principal enzyme reducing both drugs, while cytosolic aldo-keto reductase AKR1C3 or microsomal reductases probably did not play important role in metabolism of either DOX or ORC. The exposure of MCF-7 cells to low (nanomolar) concentrations of DOX or ORC caused a significant elevation of reduction rates of both cytostatics, accompanied with an increase of CBR1 protein levels. Taken together, the present results seem to suggest that the accelerated metabolic deactivation of ORC or DOX might contribute to the survival of breast cancer cells during exposure to these cytostatics.

Reference Genes for Real-Time PCR Quantification of Messenger RNAs and MicroRNAs in Mouse Model of Obesity

UNLABELLED Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of NAD(P)H quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice.

Inhibition and induction of glutathione S-transferases by flavonoids: possible pharmacological and toxicological consequences.

Many studies reviewed herein demonstrated the potency of some flavonoids to modulate the activity and/or expression of glutathione S-transferases (GSTs). Because GSTs play a crucial role in the detoxification of xenobiotics, their inhibition or induction may significantly affect metabolism and biological effects of many drugs, industrials, and environmental contaminants. The effect of flavonoids on GSTs strongly depends on flavonoid structure, concentration, period of administration, as well as on GST isoform and origin. Moreover, the results obtained in vitro are often contrary to the vivo results. Based on these facts, the revelation of important flavonoid-drug or flavonoid-pollutant interaction has been complicated. However, it should be borne in mind that ingestion of certain flavonoids in combination with drugs or pollutants (e.g., acetaminophen, simvastatin, cyclophosphamide, cisplatine, polycyclic aromatic hydrocarbons, chlorpyrifos, acrylamide, and isocyanates), which are GST substrates, could have significant pharmacological and toxicological consequences. Although reasonable consumptions of a flavonoids-rich diet (that may lead to GST induction) are mostly beneficial, the uncontrolled intake of high concentrations of certain flavonoids (e.g., quercetin and catechins) in dietary supplements (that may cause GST inhibition) may threaten human health.

Possibilities to increase the effectiveness of doxorubicin in cancer cells killing

Anthracycline antibiotic doxorubicin (DOX) belongs among the most important antineoplastics used in cancer therapy. Unfortunately, its cytostatic effect in therapeutic doses is frequently insufficient; but the use of higher DOX doses is limited by the development of systemic toxicity, especially cardiotoxicity. Therefore, a searching for some possibilities of how to increase DOX efficacy in cancer cells, and minimizing associated toxicities to noncancerous tissues, is in the forefront of scientific research. Many approaches are based on altered DOX metabolism. The classical strategies include an enhancing of DOX uptake by cancer cells and/or an activation of DOX prodrug within cancer cells via liposomal encapsulation or conjugation with antibodies, peptides, or synthetic polymers. The diminishing of DOX deactivation, restriction of DOX efflux from cancer cells, decreased antioxidant defense of cancer cells, changes in cell cycle, or modulation of signaling pathways represent newer approaches in increasing DOX toxicity in tumors. Each way has certain advantages and limitations. The aim of this review was not to collect all reported results, but to bring an overview of various approaches and a summary of their principles. Possible advantages, disadvantages, and further perspectives are discussed and evaluated.

The effects of benzimidazole anthelmintics on P4501A in rat hepatocytes and HepG2 cells.

Benzimidazole anthelmintics including albendazole, fenbendazole, and mebendazole are widely used in veterinary medicine. The effects of these benzimidazoles on cytochrome P4501A were investigated in primary cultures of rat hepatocytes and in the HepG2 cell line. After incubation of rat hepatocytes and HepG2 for 24-, 48-, and 72-h cells with drugs at various concentrations (0.1-50 microM), the enzyme activities associated with P4501A1/2 (7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation) were measured. The P4501A1/2 protein levels in both model systems were determined by Western blotting. Although all benzimidazoles provoked a significant increase of P4501A1/2 protein levels and P4501A activities, large differences in the induction response were found which was dependent on drug structure, concentration, and model system used. Based on the results, relationships between induction potency and structure of drug were demonstrated, as well as differences between the in vitro systems used. Therefore, pharmacological and toxicological consequences of cytochrome P4501A induction by benzimidazole drugs should be taken into account in veterinary therapy.

Stereochemical aspects of carbonyl reduction of the original anticancer drug oracin by mouse liver microsomes and purified 11β-hydroxysteroid dehydrogenase type 1

Oracin, 6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c] isoquinoline, is a potential cytostatic drug for oral use and presently in phase II of clinical trials. Major advantages of this novel chemotherapeutic are the possibility of oral administration, its negative results in the Ames test on mutagenicity, and the lack of cardiotoxicity. Metabolic studies on oracin have revealed that the principal metabolite in all laboratory animals is 11-dihydrooracin (DHO), which is produced by carbonyl reduction of the parent compound. Since the carbonyl moiety of oracin is a pro-chiral centre, reduction may lead to the two stereoisomer forms (+)-DHO and (-)-DHO. The aim of the present study was to infer if 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is responsible for carbonyl reduction of oracin in mouse liver and if this enzyme exhibits stereospecificity in DHO formation. 11beta-HSD 1 was purified from mouse liver microsomes, and the kinetics and stereospecificity regarding DHO formation were determined and compared to values obtained from the whole microsomal fraction. We could show that purified mouse liver 11beta-HSD 1 catalyzes the stereospecific carbonyl reduction of oracin, thereby following a sigmoidal dose-response kinetics. Due to a different ratio of (+)-DHO and (-)-DHO (93:7) formed by purified 11beta-HSD 1 compared to that produced in whole microsomes (70:30), the existence of at least one other oracin carbonyl reducing enzyme can be expected in mouse liver microsomes. This suggestion is further supported by the fact that the Hill coefficient of 2 for purified 11beta-HSD 1 (which is supporting earlier data on the cooperativity of this dimeric enzyme) changes to a Hill coefficient of 3 in whole microsomes (which is indicative for another enzyme participating in oracin carbonyl reduction).