2,4-Disubstituted pyridine derivatives are effective against intracellular and biofilm-forming tubercle bacilli.

It was recently reported that 4-substituted picolinohydrazonamides carrying hydrophilic cyclic amines, such as morpholine and pyrrolidine, at the end of their thiosemicarbazide chain have potent antimycobacterial activity in vitro at concentrations below 1 µg/ml. Here, two selected compounds, 2,4-disubstituted pyridine derivatives 11 and 15, revealed significant bactericidal activity against Mycobacterium tuberculosis localized intracellularly within human macrophages, as well as against biofilm-forming tubercle bacilli. Mutants were selected that were resistant to the investigated compounds at an efficiency similar to that identified in the presence of the first line antituberculosis drug rifampicin. The resistant mutants were viable in the presence of the tested compounds exclusively on solid media. Genome-wide sequencing of the mutants selected in the presence of compound 11 revealed the accumulation of nonsynonymous mutations in the mmpR5 gene encoding a transcriptional repressor of the MmpS5-MmpL5 efflux pump, whose upregulation has been associated with bedaquiline resistance. The depletion of MmpR5 in wild-type M. tuberculosis using CRISPR-Cas9 technology increased the resistance of this strain to compound 11. Mass spectrometry-based proteomics (LC-MS/MS) of wild-type tubercle bacilli growing in subinhibitory concentrations of compounds 11 or 15 revealed 15 overproduced proteins not detectable in the control cells, including virulence-related proteins.

[Can Analysis of Cellular Lipidome Contribute to Discrimination of Tumour and Non-tumour Colon Cells?] / Muze analýza bunecného lipidomu prispet k rozlisení nádorových a nenádorových bunek tlustého streva?

BACKGROUNDS: Colon cancer development is often characterized by abnormalities in lipid synthesis and metabolism, which may influence energetic balance, structure and function of biological membranes, or production of specific mediators and cell signalling. The changes in lipid profile and metabolism (lipidome) may significantly affect cell behaviour and response to therapy. Permanent epithelial cell lines at various stages of cancer development are used for better understanding of this topic on cellular and molecular levels. In our study, we hypothesized that detailed analyses of colon cancer cell line lipidomes may help to identify major alterations in the amount and profile of specific lipid classes/species, which can contribute to their different response to various stimuli. MATERIAL AND METHODS: Cellular lipids were isolated from six human epithelial cell lines derived from tissues at various stages of tumour development. Liquid chromatography coupled with tandem mass spectometry analyses were performed in order to determine amount and mass profiles of all phospholipid (PL), lysophospholipid (lysoPL) and sphingolipid classes. The data was statistically evaluated (cluster and discrimination analyses) with respect to mutual comparison of cell lines and to significantly discriminating lipid types. RESULTS: The results of cluster analysis arranged cell lines in order corresponding to their level of transformation (normal cells, adenoma, carcinoma and lymph node metastasis). The results of discrimination analyses revealed the most discriminating lipid types and distinction in PL: lysoPL ratios. Particularly, significant correlation of the amount and profiles of both specific lysoPL and sphingolipid classes with cell transformation level were observed. Similar approaches are now applied to compare lipidomes of colon epithelial cells isolated from tumour vs. non-tumour samples of colon cancer patients. CONCLUSION: Our results indicate that a) selected cancer cell lines are suitable model for lipidomic studies that can serve as a basis for subsequent clinical research, b) cellular lipidome analyses may help to discriminate tumour and non-tumour cells in clinical samples, where specific types of lipids could serve as biomarkers.Key words: colon cancer - cell lines - liquid chromatography - mass spektrometry - phospholipids - sphingolipids - bioinformatics The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This work was supported by Czech Health Research Council, grant No. AZV 15-30585A.Submitted: 19. 3. 2018Accepted: 18. 4. 2018.

Non-dioxin-like organic toxicant PCB153 modulates sphingolipid metabolism in liver progenitor cells: its role in Cx43-formed gap junction impairment.

The non-dioxin-like environmental toxicant 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), member of a group of persistent organic pollutants wide-spread throughout the environment, reduces gap junction intercellular communication (GJIC), an event possibly associated with tumor promotion. Since very few studies have investigated the signaling effectors and mode(s) of action of PCB153, and it is known that the gap junction (GJ) protein Cx43 can be regulated by the bioactive sphingolipid (SL) sphingosine 1-phosphate (S1P), this in vitro study mainly addresses whether SL metabolism is affected by PCB153 in rat liver epithelial WB-F344 cells. PCB153 treatment obtained significant changes in the S1P/ceramide (Cer) ratio, known to be crucial in determining cell fate. In particular, an increase in S1P at 30 min and a decrease of the bioactive lipid at 3 h were observed, whereas Cer level increased at 1 h and 24 h. Notably, a time-dependent modulation of sphingosine kinase (SphK), the enzyme responsible for S1P synthesis, and of its regulators, ERK1/2 and protein phosphatase PP2A, supports the involvement of these signaling effectors in PCB153 toxicity. Electrophysiological analyses, furthermore, indicated that the lipophilic environmental toxicant significantly reduced GJ biophysical properties, affecting both voltage-dependent (such as those formed by Cx43 and/or Cx32) and voltage-independent channels, thereby demonstrating that PCB153 may act differently on GJs formed by distinct Cx isoforms. SphK down-regulation alone induced GJIC impairment, and, when combined with PCB153, the acute effect on GJ suppression was additive. Moreover, after enzyme-specific gene silencing, the SphK1 isoform appears to be responsible for down-regulating Cx43 expression, while being the target of PCB153 at short-term exposure. In conclusion, we provide the first evidence of novel effectors in PCB153 toxic action in rat liver stem-like cells, leading us to consider SLs as potential markers for preventing GJIC deregulation and, thus, the tumorigenic action elicited by this environmental toxicant.

Phthalates deregulate cell proliferation, but not neuroendocrine transdifferentiation, in human LNCaP prostate cancer cell model.

Phthalate esters are ubiquitous environmental pollutants widely used as plasticizers, which have been shown to interfere with both endocrine regulation and development of reproductive organs. In the present study, we examined the impact of diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) on the proliferation of androgen-sensitive human prostate carcinoma LNCaP cells and related events. The results showed that both compounds were able to inhibit cell cycle progression in a dose-dependent manner. However, only DEHP was found to weakly reduce androgen receptor (AR) protein levels after long-term exposure, while only DBP partially inhibited expression of the prostate-specific antigen (KLK3) gene, a model AR transcriptional target. This indicated that inhibition of cell proliferation was likely independent of any AR modulations. Both phthalates induced suppression of cell proliferation, but none of them affected the levels of markers associated with neuroendocrine transdifferentiation (NED) in LNCaP cells. Taken together, the presented data indicate that phthalates may exert long-term negative effects on the proliferation of prostate epithelial cells derived from the carcinoma model, which are, nevertheless, largely independent of the modulation of AR expression/activity, and which do not alter further processes associated with NED.

2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) induces degradation of adherens junction proteins and inhibits beta-catenin-dependent transcription in liver epithelial cells.

The toxic modes of action of non-dioxin-like polychlorinated biphenyls (PCBs) in liver cells are still only partially understood. Several recent studies have indicated that PCBs may interfere with cell membrane protein functions. Therefore, we analyzed in the present study the effects of di-ortho-substituted 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) on proteins involved in the formation of adherens junctions in a model of rat liver progenitor cells - WB-F344 cell line. PCB 153, at micromolar concentrations, induced a gradual degradation of E-cadherin, beta-catenin or plakoglobin (gamma-catenin) proteins. This effect was not due to changes in gene expression, as PCB 153 had no effect on mRNA levels of the above mentioned proteins. Moreover, apart from the reduction of total beta-catenin pool, PCB 153 also decreased levels of the active beta-catenin form, dephosphorylated at residues Ser37 and Thr41, which is the key co-activator of Wnt-induced TCF/LEF-dependent gene expression. Therefore, we also evaluated the impact of PCB 153 on expression of Axin2, a known transcriptional target of canonical Wnt signaling. PCB 153 reduced basal Axin2 mRNA levels and it inhibited induction of Axin2 expression by recombinant mouse Wnt3a. Nevertheless, PCB 153 had no effect on phosphorylation of glycogen synthase kinase-3beta (GSK-3beta), which is supposed to target beta-catenin for its proteasomal degradation. This suggested that GSK-3beta activity is not modulated by PCB 153 and, consequently, not involved in the observed PCB 153-induced decrease of both total and active beta-catenin levels. Protein levels of E-cadherin and beta-catenin were partially restored with lysosomal inhibitor leupeptin, thus suggesting a possible role of lysosomes in the observed degradation of adherens junction proteins. Taken together, the present data suggest that PCB 153 may interfere with functions of adherens junction proteins involved in both cell-to-cell communication and intracellular signaling. Such mechanisms might be involved in the effects of non-dioxin-like PCBs contributing to liver tumor promotion.

Effects of methylated chrysenes on AhR-dependent and -independent toxic events in rat liver epithelial cells.

Methylated chrysenes (MeChry) are important cigarette smoke constituents and 5-MeChry has been listed as possibly carcinogenic to humans. Although a major attention has been in past paid especially to mutagenic, tumor-initiating effects of MeChry, little is known about toxic effects of MeChry related to tumor promotion. As the position of methyl group has been repeatedly observed to determine genotoxic effects of MeChry, we examined both genotoxic and nongenotoxic effects of MeChry, using rat liver cell lines as experimental models. All six MeChry were relatively efficient aryl hydrocarbon receptor (AhR) agonists, with 3- and 6-MeChry being the most potent inducers of the AhR-mediated reporter gene activity. All six compounds disrupted contact inhibition in rat liver epithelial WB-F344 cells, a process previously reported to be AhR-dependent, suggesting that MeChry may interfere with cell cycle control in an AhR-dependent manner. In contrast, only 5- and 6-MeChry were found to acutely inhibit gap junctional intercellular communication (GJIC), another parameter correlating with tumor promoting effects of xenobiotics. Both 5- and 6-MeChry were efficient inducers of mRNA expression of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons, including cytochromes P450 1A1/1B1 and aldo-keto reductase 1C9. However, only 5-MeChry, and not 6-MeChry, induced significant formation of DNA adducts in rat liver epithelial cells, which corresponded with its ability to induce high accumulation of cells in S-phase. On the other hand, 5-MeChry induced neither apoptosis related to DNA damage nor phosphorylation of p53 tumor suppressor. Taken together, our results suggest that methyl group position may affect both genotoxic and nongenotoxic effects of MeChry, such as formation of DNA adducts and inhibition of GJIC. All MeChry showed a potency to disrupt cell proliferation control, while 5-MeChry was a single compound inducing DNA damage, disruption of cell cycle control and inhibition of GJIC in rat liver cells.

Non-dioxin-like polychlorinated biphenyls induce a release of arachidonic acid in liver epithelial cells: a partial role of cytosolic phospholipase A(2) and extracellular signal-regulated kinases 1/2 signalling.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) have been shown to act as tumor promoters in liver; however, the exact mechanisms of their action are still only partially understood. One of the interesting effects of NDL-PCBs is the acute inhibition of gap junctional intercellular communication (GJIC), an effect, which has been often found to be associated with tumor promotion. As previous studies have suggested that NDL-PCB-induced disruption of lipid signalling pathways might correspond with GJIC inhibition, we investigated effects of PCBs on the release of arachidonic acid (AA) in the rat liver epithelial WB-F344 cell line, a well-established model of liver progenitor cells. We found that both 2,2',4,4'-tetrachlorobiphenyl (PCB 47) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), but not the dioxin-like, non-ortho-substituted, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), induce a massive release of AA. The AA release, induced by PCB 153, was partially inhibited by extracellular signal-regulated kinases 1/2 (ERK1/2) signalling inhibitor, U0126, and by cytosolic phospholipase A(2) (cPLA(2)) inhibitor, AACOCF(3). Although PCB 153 induced both ERK1/2 and p38 activation, the specific p38 kinase inhibitor, SB203580, had no effect on AA release. Inhibitors of other phospholipases, including phosphatidylcholine-specific phospholipase C or phosphatidylinositol-specific phospholipase C, were also without effect. Taken together, our findings suggest that the AA release, induced by non-dioxin-like PCBs in liver progenitor cell line, is partially mediated by cytosolic PLA(2) and regulated by ERK1/2 kinases. Our results suggest that more attention should be paid to cell signalling pathways regulated by AA or eicosanoids after PCB exposure, which might be involved in their toxic effects.

Dibenzanthracenes and benzochrysenes elicit both genotoxic and nongenotoxic events in rat liver 'stem-like' cells.

Polycyclic aromatic hydrocarbons (PAHs) with molecular weight 278 are a group of PAHs that are mostly not covered by the current monitoring programs, despite their relative abundance in environmental samples and possible carcinogenicity. Although benzo[g]chrysene (BgChry) and dibenz[a,h]anthracene (DBahA) have been for a long time studied as genotoxic, tumour-initiating compounds, little is known about the potential tumour-promoting effects of this group of PAHs. In the present study, we investigated their impact on activation of the aryl hydrocarbon receptor (AhR), induction of enzymes involved in metabolic activation of PAHs, disruption of cell cycle control in confluent cell population and inhibition of gap junctional intercellular communication (GJIC), using the rat liver epithelial cell line WB-F344 as a model of liver progenitor cells. We found that BgChry was the weakest inducer of the AhR-mediated activity, while relative potencies of benzo[b]chrysene (BbChry) and benzo[c]chrysene (BcChry) were comparable to the previously reported values for dibenzanthracenes. All compounds increased expression of cytochromes P450 1A1 and 1B1, and aldo-keto reductase 1C9. BgChry was found to induce high amounts of DNA adducts, which corresponded with induction of p53 phosphorylation at Ser15, apoptosis and accumulation of cells in S-phase of cell cycle, leading to a decrease in cell numbers. All other compounds were found to stimulate cell proliferation in contact-inhibited WB-F344 cells in a dose-dependent manner. We found that only BgChry, and to a lesser extent also BcChry, inhibited GJIC at high concentrations. Taken together, dibenzanthracenes and benzochrysenes, with exception of BgChry, seem to act primarily through deregulation of cell proliferation in liver epithelial cells, which is related to their relatively high AhR-mediated activity. The disruption of cell cycle control might contribute to their carcinogenic effects, as well as to carcinogenicity of complex environmental mixtures containing high levels of PAHs with molecular weight 278.

Immunomodulating effect of influenza vaccination in the elderly differing in health status.

The aim of this study was to analyse whether split influenza vaccine may elicit NK cytotoxic response in the vaccinated elderly people and whether this effect may be maintained over few weeks after vaccination. It was also worth investigating the relation between NK activity in the vaccinated and specific immune protection against influenza and non-specific against other infections. Two groups of volunteers were vaccinated with trivalent split viron influenza vaccine in two consecutive seasons (1999/2000; 2000/2001). The elderly group consisted of 142 people (65-92 years old) in the first season and 110 in the second; while the young (16-44 years old) of 98 and 67 people, respectively. An analysis of NK cytotoxic activity had been done before vaccination, two days, one month and fifth months thereafter. The results revealed that vaccination with the influenza vaccine had an augmenting effect on NK activity, in all groups examined, in both epidemic seasons, visible at two days and 1 month after the vaccination. In the elderly high pre- and post-vaccination NK activity was related to higher titers of anti-hemagglutinin, better health status and lower incidence of all cause respiratory tract infections. At the second vaccination, most of the elderly with chronic medical conditions and high NK activity, who did not attain the protective level of anti-hemagglutinins in the first season, converted into the protected. High pre- and post-vaccination NK activity predisposes elderly people to the protective humoral anti-hemagglutinin response and gives better protection from respiratory tract infections.

A combined chemical and bioassay analysis of traffic-emitted polycyclic aromatic hydrocarbons.

The objective of this study was to determine the concentrations of an extended series of polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs in outdoor air samples collected in low-contaminated urban areas, affected mainly by traffic emissions, and to estimate in vitro mutagenic and dioxin-like toxicity of extracts from these samples. Data on concentrations of PAHs and toxic in vitro potencies were compared in extracts obtained by different sampling methods. PAHs and their derivatives were analysed by high performance liquid chromatography with diode array and fluorescence detection, as well as gas chromatography with mass spectrometry. The total sum of 39 PAHs under study ranged from 6.7 to 62.7 ng.m(-3); of this, the sum of 16 U.S. EPA priority PAHs in urban air samples ranged from 3.2 to 6.2 ng.m(-3). Phenanthrene was the prevalent PAH in all air samples tested, with concentrations up to 17.6 ng.m(-3), followed by fluorene, fluoranthene and pyrene present mostly in the gaseous phase. Also, other low molecular weight PAHs (with MW up to 228) were distributed mostly in gaseous phase. The particulate phase contained mostly carcinogenic PAHs, among which, benzo[a]pyrene, indeno[1,2,3-cd]pyrene and benzofluoranthenes were predominant compounds (with benzo[a]pyrene reaching levels up to 1.57 ng.m(-3)). Traffic emissions were confirmed as the major source of PAHs in the airborne samples due to the presence of elevated concentrations of benzo[ghi]perylene and coronene. The most abundant nitrated PAH derivatives were nitronaphthalenes, which were present exclusively in the vapor phase; 9-nitroanthracene, 9-nitrophenantrene and 3-nitrofluoranthene were associated mostly with particulate matter (PM(10)). Bioassays for detection of the Ah receptor-mediated activity and mutagenicity in vitro were used as a screen of potential adverse effects of air pollutants emitted from traffic. The major part of mutagenic and aryl hydrocarbon receptor (AhR)-mediated activities was found to be present in the PM(10) fraction. Although the PM(10) sampling technique was found to be a suitable method regarding the subsequent determination of mutagenic and AhR-mediated activities in vitro, relative toxic potencies, associated with low molecular weight PAHs (such as tumor promotion and other adverse effects), could be underestimated.

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.

The role of Bak expression in apoptosis of the oral squamous cell carcinoma (OSCC) and metastases to lymph nodes (LNMs).

The immunohistochemical method was applied to show Bak expression in oral squamous cell carcinoma and its metastases to lymph nodes (LNMs). Bak expression was evaluated by immunohistochemical methods in specimens with oral squamous cell carcinomas and their lymph node metastases. Immunohistochemical studies were performed, using goat polyclonal Bak antibodies (Santa Cruz Biotechnology, USA) at 1:200 dilution. Our studies revealed over expression (64%) of Bak in the cytoplasm of epithelial cells in primary tumours (PTs) and in (75%) LNMs. No statistically significant correlations were observed between Bak immunoreactivity and age, pT and G of the carcinoma in PTs and LNMs. We conclude that expression of Bak may be useful for better characterising and predicting the prognosis of OSCC but cooperative studies are needed to assess its applications in the clinical practice.

C-erb-B2 and Bcl-xl protein expression in Barrett's oesophagus in correlation with morphological parameters.

The aim of the study was to evaluate the correlation of c-erb-b2 and Bcl-xl expression in biopsy specimens of Barrett's oesophagus from 44 patients with morphological features. The examined group was subdivided into: negative for dysplasia, indefinite for dysplasia, positive for dysplasia-low grade, and adenocarcinoma with high grade dysplasia. Positive c-erb-B2 staining was found in 34.1% and Bcl-xl protein expression was observed in 96.9% of BE. The results showed increased c-erb-B2 and Bcl-xl protein expressions with progressive grades of dysplasia to adenocarcinoma. In conclusion, an evaluation of c-erb-B2 and Bcl-xl expression can be useful for the histopatologic diagnosis of BE and correct interpretation of dysplasia.

Proliferating activity in the epithelial and stromal component of fibroadenomas and phyllodes tumours of the breast.

The aim of the study was an evaluation of PCNA and Ki-67 expression in the epithelial and stromal component of fibroepithelial tumours (FT) of the breast in correlation with morphological parameters. A series of 11 fibroadenomas (FA), including 8 cases of the cellular type (FAC), 19 benign phyllodes tumours (PTLGM), 8 bordeline (PTBM) and 6 malignant phyllodes tumours were assessed, using immunohistochemistry. The expressions of Ki-67 and PCNA in the epithelial component were significantly higher in PTLGM, when compared with FA and PTBM. A significant increase of Ki-67 and PCNA stromal expressions was associated with the progression from PTLGM to PTHGM. Our results show that Ki-67 and PCNA may be useful in the evaluation of stromal proliferation in phyllodes tumours (PT), which play an integral part in the progression from PTLGM through PTBM to PTHGM.

Influence of doxycycline on the epiphyseal plate cartilage of the rats in experimental osteoarthrosis, induced by iodoacetate.

In 36 Wistar rats with the iodoacetate-induced experimental osteoarthrosis (OA), effects of doxycycline, given orally, were determined on histochemical reactions of glycosaminoglycans (GAG) in the epiphyseal plate cartilage. The epiphyseal plate of rats with OA was reduced in height (especially the proliferative zone), cell columns were disorganized, many chondrocytes were irregular and polygonal, their nuclei were pycnotic, the intensity of GAG staining was irregular and predominantly reduced, which can be interpreted as signs of degeneration. A concomitant administration of doxycycline in the second group of rats prevented, to some extent, the negative effects of iodoacetate on chondrocytes and led to a more pronounced intensity of GAG reactions in the matrix of the epiphyseal plate.

Cell bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in environmental samples.

In vitro cell bioassays are useful techniques for the determination of receptor-mediated activities in environmental samples containing complex mixtures of contaminants. The cell bioassays determine contamination by pollutants that act through specific modes of action. This article presents strategies for the evaluation of aryl hydrocarbon receptor (hereafter referred as dioxin-like) or estrogen receptor mediated activities of potential endocrine disrupting compounds in complex environmental mixtures. Extracts from various types of environmental or food matrices can be tested by this technique to evaluate their 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents or estrogenic equivalents and to identify contaminated samples that need further investigation using resource-intensive instrumental analyses. Fractionation of sample extracts exhibiting significant activities, and subsequent reanalysis with the bioassays can identify important classes of contaminants that are responsible for the observed activity. Effect-directed chemical analysis is performed only for the active fractions to determine the responsible compounds. Potency-balance estimates of all major compounds contributing to the observed effects can be calculated to determine if all of the activity has been identified, and to assess the potential for interactions such as synergism or antagonism among contaminants present in the complex mixtures. The bioassay approach is an efficient (fast and cost effective) screening system to identify the samples of interest and to provide basic information for further analysis and risk evaluation.

Enhancement of beta-sitosterol transformation in Mycobacterium vaccae with increased cell wall permeability.

Mycobacterium vaccae exposed to compounds which are known to disorganise the cell wall composition and architecture (protamine, glycine) showed increased specific activity in beta-sitosterol biotransformation to androstene derivatives, intennediates in the production of most medical steroids. GC/MS analysis of free lipid fatty acids revealed higher content of unsaturated compounds, mainly C16:1 and C18:1 in protamine- and glycine-treated cells than that in control cells, which seems to change the permeability features of the cell wall barrier, facilitating hydrophobic beta-sitosterol diffusion.

Effect of ivermectin on activities of cytochrome P450 isoenzymes in mouflon (Ovis musimon) and fallow deer (Dama dama).

Ivermectin is an antiparasitic drug widely used in veterinary and human medicine. We have found earlier that repeated treatments of rats with high doses of this drug led to significant increase of cytochrome P450-dependent 7-methoxyresorufin O-demethylase (MROD) and 7-ethoxyresorufin O-deethylase (EROD) activities in hepatic microsomes. In the present study, the effects of ivermectin on cytochrome P450 (CYP) activities were investigated in mouflon (Ovis musimon) and fallow deer (Dama dama). This study was conducted also to point out general lack of information on both basal levels of CYP enzymes and their inducibilities by veterinary drugs in wild ruminants. Liver microsomes were prepared from control animals, mouflons, after single or repeated (six doses in six consecutive days) treatments with therapeutic doses of ivermectin (0.5 mg kg(-1) of body weight), and fallow deer exposed to repeated doses of ivermectin under the same conditions. Alkyloxyresorufins, testosterone and chlorzoxazone were used as the specific substrate probes of activities of the CYP isoenzymes. A single therapeutic dose of ivermectin significantly induced (300-400% of the control group) the activities of all alkyloxyresorufin dealkylases tested in mouflon liver microsomes. Repeated doses of ivermectin also caused an increase of these activities, but due to fair inter-individual differences, this increase was not significant. The administration of ivermectin led to an induction (170-210% of the control) of the testosterone 6beta- and 16alpha-hydroxylase activities in mouflon liver but no significant modulation of chlorzoxazone hydroxylase (CZXOH) activity was found in mouflon liver. CYP-dependent activities in hepatic microsomes were generally higher in fallow deer than in mouflons. However, with the exception of slight increase in the 7-benzyloxyresorufin O-dealkylase (BROD) activities, no significant modulation of the other activities was observed. The induction of CYP3A-like isoenzyme was confirmed by immunoblotting only in the microsomes from mouflons administered with repeated doses of ivermectin; however, no significant increase of CYP1A isoenzymes was observed due to a weak cross-reactivity of anti-rat CYP1A1/2 polyclonal antibodies used in the study. The results indicate that ivermectin should be considered as an inducer of several cytochrome P450 isoenzymes, including CYP1A, 2B and 3A subfamilies, in mouflons. The comparison of induction effect of ivermectin in rat, mouflon and fallow deer also demonstrates the inter-species differences in inducibility of CYP enzymes.

[Influenza in Poland in 1999]. / Grypa w 1999 roku.

A total of 2,344,773 cases of influenza were reported in Poland in 1999, corresponding to 6066.1 cases per 100,000 population and was 2.8 times higher as compared with 1998. The highest influenza incidence rate (10,770.9 per 100,000) was reported in Malopolskie voivodeship. Children up to 14 years old accounted for 34.9% (818,629; incidence 10,616.2 per 100,000) of all reported influenza cases. A total of 3,925 persons required hospitalization, eight times more as compared with 1998. The number of deaths due to influenza amounted to 402. Compared with 1998, in 1999 reported influenza deaths increased 6.4 times. During the second two weeks of January 1999 four influenza strains of type B were isolated from patients aged 13, 16, 31, and 38. During the first part of February 1999 another two strains of influenza virus type B were obtained from persons aged 35 and 45. All six isolates were related antigenically to the vaccine strain B/Beijing/184/93 and were confirmed by the WHO Collaborating Center for Influenza Reference and Research, London.

Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay.

Activation of aryl hydrocarbon receptor (AhR) by 30 polycyclic aromatic hydrocarbons (PAHs) was determined in the chemical-activated luciferase expression (CALUX) assay, using two exposure times (6 and 24h), in order to reflect the metabolization of PAHs. AhR-inducing potencies of PAHs were expressed as induction equivalency factors (IEFs) relative to benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 24h exposure assay, the highest IEFs were found for benzo[k]fluoranthene, dibenzo[a,h]anthracene and dibenzo[a,k]fluoranthene (approximately three orders of magnitude lower than TCDD) followed by dibenzo[a,j]anthracene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene, and naphtho[2,3-a]pyrene. The 6h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24h exposure (generally by two orders of magnitude), probably due to the high rate of PAH metabolism. The strongest AhR inducers showed IEFs approaching that of TCDD. Several PAHs, including some strong mutagens, such as dibenzo[a,l]pyrene, cyclopenta[cd]pyrene, and benzo[a]perylene, elicited only partial agonist activity. Calculation of IEFs based on EC25 values and/or 6h exposure data is suggested as an alternative approach to estimation of toxic potencies of PAHs with high metabolic rates and/or the weak AhR agonists. The IEFs, together with the recently reported relative mutagenic potencies of PAHs [Mutat. Res. 371 (1996) 123; Mutat. Res. 446 (1999) 1] were combined with data on concentrations of PAHs in extracts of model environmental samples (river sediments) to calculate AhR-mediated induction equivalents and mutagenic equivalents. The highest AhR-mediated induction equivalents were found for benzo[k]fluoranthene and benzo[j]fluoranthene, followed by indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[a]pyrene, dibenzo[a,j]anthracene, chrysene, and benzo[b]fluoranthene. High mutagenic equivalents in the river sediments were found for benzo[a]pyrene, dibenzo[a,e]pyrene, and naphtho[2,3-a]pyrene and to a lesser extent also for benzo[a]anthracene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[j]fluoranthene, dibenzo[a,e]fluoranthene and dibenzo[a,i]pyrene. These data illustrate that AhR-mediated activity of PAHs, including the highly mutagenic compounds, occurring in the environment but not routinely monitored, could significantly contribute to their adverse effects.