Maciej Góra

Microcystin-LR induced apoptosis and mRNA expression of p53 and cdkn1a in liver of whitefish (Coregonus lavaretus L.).

There is growing evidence that adverse effects of microcystin-LR (MC-LR) are closely related to oxidative stress processes, free radicals and DNA damage, and involve major gene transcript changes. This study, utilizing gene expression analysis and plasma chemistries was the first to measure the effects of MC-LR in whitefish (Coregonus lavaretus L.), a feasible organism for pollution monitoring in aquatic systems. Fish were injected with different concentrations of MC-LR (0, 10 and 100 microg/kg of body weight) and then sacrificed at either 0, 8, 24, 48 or 72 h later, and their liver tissue were harvested for detailed investigation. Specifically, we were interested whether MC-LR is capable of: (i) modulating expression of two genes, tumor suppressor gene p53 and cdkn1a, p53 direct transcription target, and (ii) inducing apoptosis in whitefish liver. To study these effects, we developed a real-time qPCR assays useful for measuring both p53 and cdkn1a gene transcript levels in liver. To obtain necessary information for the study, either full-length p53 cDNA of whitefish (Wf-p53) was determined, using molecular cloning and rapid amplification of cDNA ends (RACE), or as for Wf-cdkn1a, specific primers were designed based on highly conserved regions of cdkn1a in fish. The Wf-p53 was found to share the same characteristics with a known p53 mRNA sequence of other vertebrates. Whitefish p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. The injection study showed that MC-LR at a higher dose, i.e. 100 microg/kg body weight, up-regulated expression of p53 and cdkn1a genes in whitefish liver, as reflected by the continuous increase in their mRNA levels through the whole experiment. Furthermore, DNA fragmentation was observed in liver cells of whitefish after 24h of exposure to MC-LR (100 microg/kg) that suggests the possibility of apoptosis. Finally, the study confirmed previous observations of severe injury of the liver and loss of normal organ functions as revealed by elevated levels of blood AspAT, AlaAT, and hepatosomatic index (HSI).

Effects of cyclopenta[c]phenanthrene and its derivatives on zona radiata protein, ERα, and CYP1A mRNA expression in liver of rainbow trout (Oncorhynchus mykiss Walbaum)

Despite cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) have been detected in the environment, the ability of CP-PAH to induce cellular and tissue responses remains poorly characterized. In this study, xenoestrogen-associated responses (mRNA levels of estrogen receptor alpha, ERalpha, and zona radiata protein, Zrp) and xenobiotic effects (CYP1A mRNA) have been investigated in liver of juvenile rainbow trout after short-term treatment (8 and 24 h) with following compounds administered singly: cyclopenta[c]phenanthrene (CP[c]Ph); its derivatives, 5A-CP[c]Ph; 5A6M-CP[c]Ph; 5A9M-CP[c]Ph; B[c]Ph, a structurally similar polycyclic aromatic hydrocarbon; B[a]P, a model CYP1A inducer; and zearalenone (ZEA), naturally occurring ligand for ER. The CYP1A mRNA expression after 24 h of exposure with CP[c]Ph or its derivatives, except 5A9M-CP[c]Ph, was 3-9-fold higher compared to controls (P<0.05), but it was less than that caused by B[a]P (65-fold up regulation; P<0.01). Moreover, neither of the CP-PAH compounds modulated liver ERalpha or Zrp mRNA levels as compared to effects associated with ZEA. Interestingly, a treatment with this ER-ligand, caused moderate but significant increase of CYP1A mRNA expression (about 2.5-fold; P<0.05). The finding that ZEA is capable of acting as either estrogenic and xenobiotic compound, should be further explored in a more detailed and differently designed experiment.

LIF excitation spectra of 2,6-dicyanoaniline

Abstract 2,6-Dicyanoaniline (DCA) reveals remarkably different physicochemical and spectral properties than other aniline derivatives. The infrared and Raman spectra for DCA in solid state and laser induced fluorescence (LIF) excitation spectra of vacuum jet cooled molecules have been investigated. The ground S 0 and the S 1 (B 2 ) excited state geometries, charge distributions and vibrational frequencies of DCA have been evaluated by ab initio calculations. The Dushinsky matrices have been applied for the mode correlation and assignments of the transition frequencies in LIF excitation spectra of DCA(NH 2 ) and DCA(ND 2 ). The intensity distributions in LIF excitation spectra were modeled. The dominant vibronic bands in the excitation spectra are connected with motions of the adjacent cyano- and amino groups. The transitions involving inversion motion of the amino group have low intensities.

Differential gene expression in rainbow trout (Oncorhynchus mykiss) liver and ovary after exposure to zearalenone.

Zearalenone (ZEA) is a mycotoxin of worldwide occurrence, and it has been shown to produce numerous adverse effects in both laboratory and domestic animals. However, regardless of recent achievements, the molecular mechanisms underlying ZEA toxicity remain elusive, and little is known about transcriptome changes of fish cells in response to ZEA occurrence. In the present study, differential display PCR was used to generate a unique cDNA fingerprint of differentially expressed transcripts in the liver and ovary of juvenile rainbow trout after either 24, 72, or 168 h of intraperitoneal exposure to ZEA (10 mg/kg of body mass). From a total of 59 isolated cDNA bands (ESTs), 5 could be confirmed with Real-Time qPCR and their nucleotide sequences were identified as mRNAs of: acty (β-centractin), the cytoskeleton structural element; bccip, responsible for DNA repair and cell cycle control; enoa (α-enolase), encoding enzyme of the glycolysis process; proc (protein C), that takes part in the blood coagulation process; and frih, encoding the heavy chain of ferritin, the protein complex important for iron storage. Further qPCR analysis of the confirmed ESTs expression profiles revealed significant mRNA level alterations in both tissues of exposed fish during the 168 h study. The results revealed a complex network of genes associated with different biological processes that may be engaged in the cellular response to ZEA exposure, i.e. blood coagulation or iron-storage processes.

Solvent-free condensations of ketones with malononitrile catalysed by methanesulfonic acid/morpholine system

The preparation of ylidenemalononitriles via Knoevenagel condensations of ketones with malononitrile under solvent-free conditions is described. Good yields and short reaction time are the features observed with methanesulfonic acid (MSA)/morpholine used as the catalyst. The wide applicability of the protocol is shown by the fact that not only unconjugated, but also aryl-alkyl ketones gave satisfactory yields.

CYP1A expression in liver and gills of roach (Rutilus rutilus) after waterborne exposure to two phenanthrene derivatives, 1-methylphenanthrene and 4-methylphenanthrene

Phenanthrenes (Phs) substituted with alkyl groups are a class of compound present in the environment, and they appear to be toxic to developing fish. The present study aimed to investigate the effect of waterborne exposure to two monomethyl derivatives of phenanthrene, 1-methylphenanthrene (1M-Ph) and 4-methylphenanthrene (4M-Ph), on cytochrome P450 1A (CYP1A) gene expression in fish gills and liver. Juvenile common roaches (Rutilus rutilus) were exposed to water with dimethyl sulfoxide (DMSO) solutions of 1M-Ph, 4M-Ph, benzo[a]pyrene (BaP; positive control), each at a dose of 100 µg/L, or to water with DMSO alone (negative control group) for 2 d and 7 d. Significant CYP1A responses with regard to treatment and exposure duration were noted (2-way analysis of variance [ANOVA]) in gills (p = 0.013 and p = 0.003, respectively) and liver (p < 0.001). The 2 monomethyl Phs did not induce consistent gene expression changes, except for 4-MPh, which elevated the CYP1A messenger ribonucleic acid (mRNA) level in the liver at the end of the treatment (almost 4-fold; p < 0.05; 7 d). As was expected, exposure to BaP resulted in elevation of CYP1A mRNA expression in treated fish compared with the control group. Expressions after 2 d and 7 d were approximately 220- and 180-fold higher in liver and 8- and 6-fold higher in gills respectively. The CYP1A protein levels remained stable in both tissues, with one notable exception in roach liver treated for 2 d with BaP (∼ 6-fold increase; p < 0.05). The different effects of the 1- and 4-methylphenanthrenes on CYP1A gene expression in roach liver suggest a relationship between chemical or 3-D structure of the differentially substituted monomethyl Phs and their biological activity.

CYP1A expression in liver and gills of rainbow trout (Oncorhynchus mykiss) after short-term exposure to dibenzothiophene (DBT)

Dibenzothiophene (DBT), a common component of crude oil, is a widespread environmental pollutant of known adverse effects to aquatic vertebrates. However, the molecular mechanism by which DBT exerts its effects still remains unknown. Our goal for this study was to examine DBT effects on CYP1A expression in liver and gills of rainbow trout after short-term exposure. Juvenile trout individuals were injected intraperitoneally with two doses of DBT (10 or 50mgkg(-1)) and were kept in tanks for 8 and 24h (T=14 degrees C), then their gene expression levels were evaluated by Real-Time qPCR and Western-blot analysis. Treatment with DBT at either dose decreased CYP1A mRNA levels through the exposure period, which resulted in the final decrease of CYP1A protein levels in liver and gills on the end of experiment (24h). Thus, our results showing significant depletion of CYP1A molecules in metabolic tissues upon DBT treatment correlate with those previous reports that indicate a role of DBT in reducing CYP1A activity in fish.

MOLECULAR GEOMETRY, CYP1A GENE INDUCTION AND CLASTOGENIC ACTIVITY OF CYCLOPENTA[c]PHENANTHRENE IN RAINBOW TROUT

Cyclopenta[c]phenanthrene (CP[c]Ph) is a PAH member that shows similarities to bay-and fjord region possessing PAHs. On the basis of X-ray measurements it was found that the molecule of this hydrocarbon is planar. In this case, intramolecular strains caused by repulsion between protons in the pseudo fjord-region are balanced by both the shortening of some bonds which acquire more double bond character, and by the enlargement of exocyclic angles within the pseudo fjord-region. The activity of CP[c]Ph was investigated in vivo in rainbow trout, Oncorhynchus mykiss. The CYP1A mRNA levels following 48h-treatment with CP[c]Ph or benzo[a]pyrene (B[a]P; positive control) were determined and compared with incidences of clastogenic changes observed in the peripheral blood erythrocytes. We have found that the ability to induce CYP1A by these PAH compounds is positively correlated with the incidences of clastogenic changes in rainbow trout erythrocytes.

Novel synthesis of α-arylnaphthalenes from diphenylacetaldehydes and 1,1-diphenylacetones

Abstract A two-step synthesis of 1-amino-4-arylnaphthalene-2-carbonitriles from diphenylacetaldehydes and 1,1-diphenylacetones involves condensation of the carbonyl compounds with malonodinitrile and cyclization of the aryl-ylidenemalonodinitriles obtained in concentrated sulfuric acid. The benzannulation reaction is accompanied with a quasi-aromatic rearrangement. Some of synthesized aminonitriles reveal considerable biological activity against phytopathogenic fungi.

Inactivation of Jack Bean Urease by Allicin

Allicin—diallyl thiosulfinate—is the main biologically active component of freshly crushed garlic. Allicin was synthesized as described elsewhere and was tested for its inhibitory ability against jack bean urease in 20 mM phosphate buffer, pH 7.0 at 22°C. The results indicate that allicin is an enzymatic inactivator. The loss of urease activity was irreversible, time- and concentration dependent and the kinetics of the inactivation was biphasic; each phase, obeyed pseudo-first-order kinetics. The rate constants for inactivation were measured for the fast and slow phases and for several concentrations of allicin. Thiol reagents, and competitive inhibitor (boric acid) protected the enzyme from loss of enzymatic activity. The studies demonstrate that urease inactivation results from the reaction between allicin and the SH-group, situated in the urease active site (Cys592).