Drug metabolism enzymes in a steatotic model of rat treated with a high fat diet and a low dose of streptozotocin.

Herein we have characterized CYPs and antioxidant enzymes in a new steatotic rat model induced with a high fat diet (HFD) combined with a low dose of streptozotocin (STZ). This model was recently put forward in order to better replicate the NAFLD human pathology. HFD/STZ rats developed hyperglycemia, hypercholesterolemia and overt steatosis. The treatment also caused liver damage, but not lipid peroxidation, suggesting this damage was due to hepatic fat deposition and excess formation of toxic free fatty acids, rather than to oxidative stress. In the HFD/STZ group, a significant rise in total CYP content was found, in conjunction with increased activity and protein levels of CYP2E1 and CYP4A, the latter also up-regulated at the transcriptional level. A significant decrease of CYP2C11 was observed at the transcriptional and protein level, whereas CYP3A2 did not change in response to HFD/STZ treatment. In our experimental conditions, the activity of the HO-1 and NQO1 enzymes, whose genes are regulated by Nrf2, were not affected, and nor were the antioxidant enzymes SOD and CAT, confirming the lack of oxidative stress. Our HFD/STZ treatment, which established overt steatosis and changes in CYPs expression, but not oxidative stress, likely reflects an early stage of NAFLD.

Structural influence of isothiocyanates on expression of cytochrome P450, phase II enzymes, and activation of Nrf2 in primary rat hepatocytes.

Primary cultures of rat hepatocytes were used to investigate whether and how eight isothiocynates (ITCs) with different chemical structures (the aromatic benzyl, 4-hydroxybenzyl, phenethyl isothiocyanates and the aliphatic allyl, napin, iberin, raphasatin isothiocyanates and sulforaphane) derived from hydrolyzed glucosinolates, were able to modulate cytochrome P450 (CYP) and antioxidant/detoxifying enzymes and to activate the Nrf2 transcription factor. The aromatic ITCs at 40 µM markedly increased the transcription of CYP1A1 and 1A2 mRNA and increased the associated ethoxyresorufin O-deethylase (EROD) activity after 24 h of treatment. By contrast, the aliphatic ITCs (40 µM) decreased CYP1A1 and 1A2 transcription, together with the corresponding EROD activity. The same treatment also caused a striking and similar transcriptional repression of CYP3A2, and the corresponding benzyloxyquinoline debenzylase activity in response to all the ITCs tested. In the same culture conditions, most of the antioxidant/detoxifying enzymes were significantly up-regulated by 40µM ITCs. In particular, NAD(P)H:quinone oxidoreductase and heme oxygenase-1 were induced, although to different levels, at transcriptional, protein and/or activity levels by all the ITCs. However, glutathione S-transferase activity was not induced by the allyl, benzyl, and 4-hydroxybenzyl ITCs, glutathione reductase activity was not induced by benzyl, and 4-hydroxybenzyl ITCs and catalase activity was not induced by allyl ITC. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus was revealed by immunoblotting after 1h of treatment for all the ITCs tested. The ability of ITCs to induce the antioxidant and phase II enzymes did not appear to be affected by their hydrophilicity or other structural factors. Taken together, these results show that these ITCs are effective inducers of ARE/Nrf2-regulated antioxidant/detoxifying genes and have the potential to inhibit, at least in rat liver, the bioactivation of carcinogens dependent on CYP3A2 catalysis.

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up.

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.

Protective effect of lipoic acid against hydrogen peroxide in yeast cells.

Lipoic acid (LA) is found in all kinds of cells, it is widely used in medicine and as a dietary supplement, and it is involved in different physiological functions. Even if there are many papers regarding therapeutic effects of LA, medical research does not always support its effectiveness and little is known about LA metabolism in eukaryotic cells. In this work the probable protective effect of LA was investigated employing five strains of yeast Saccharomyces cerevisiae through short term assays. In particular LA behaviour in oxidative stress conditions was studied. For this purpose hydrogen peroxide was used as oxidant. In D7 strain, LA showed antimutagenic effects against hydrogen peroxide and decreased significantly cytochrome P450. To better elucidate the effect of LA the following yeast strains carrying deletions in superoxide dismutase genes (SOD) were employed: EG-103 (wild type), EG-110 strain (without mytochondrial SOD), EG-118 (without cytoplasmatic SOD) and EG-133 (without both enzymes). LA increased the number of mitotic divisions in EG-103, EG-110 and EG-133 and in growing cells (EG-103, EG-110, EG-118) it increased survival percentage with respect to hydrogen peroxide. The positive action was evident in D7 and in EG strains and it showed that LA can be protective and antimutagenic against oxidants in yeast cells, via its antioxidant activity.

Genotoxic and mono-oxygenase system effects of the fungicide maneb.

The in vivo effects of a commercial preparation of maneb on mono-oxygenase activities of hepatic microsomes of basal and induced rats were examined. In vitro experiments with the D7 strain of yeast Saccharomyces cerevisiae were also performed. In both basal and induced rats maneb caused a decrease in cytochrome P-450 content and aniline hydroxylase. Immunoblotting analysis using anti-P-450 IIE1 antibodies confirmed the data obtained for aniline hydroxylase activity. Maneb was toxic in cells of S. cerevisiae. On the basis of in vivo and in vitro experiments it can be concluded that maneb possesses a toxic activity attributable to its main metabolite ethylene thiourea. Immunoblotting analysis indicates that maneb biotransformation influences the IIEI P-450 isoform.

Isolation of a novel metabolizing system enriched in phase-II enzymes for short-term genotoxicity bioassays.

Murine S9 liver fractions isolated from mice fed 7.5 g kg-1 2(3)-tert-Butyl-4-hydroxyanisole (BHA) for 3 weeks were tested to determine: (a) the profile of both phase-I and phase-II xenobiotic metabolizing enzymes; (b) their ability to induce in vitro covalent binding of some precarcinogens to calf thymus DNA; and (c) their activation in a standard genetic toxicology assay. With regard to phase-I pathway, the S9 fraction expressed various cytochrome P-450-(CYP) (classes 1A1, 1A2, 2B1, 2E1, and 3A)-dependent biotransformation enzymes at levels comparable with those present in murine control liver. For post-oxidative enzymes, the S9 expressed high levels of glutathione S-transferases (up to 12-fold increase), glutathione S-epoxide-transferase (up to 2.6-fold), UDP-glucuronosyl transferase (up to 5.3-fold) and epoxide hydrolase (up to 2.6-fold) activities, as compared to untreated mice. The in vitro DNA binding of the precarcinogenic agents [14C]-1,4-dichlorobenzene, [14C]-1,2-dichlorobenzene and [14C]-1,4-dibromobenzene, mediated by BHA-induced cytosol and/or microsomal preparation, showed an increase in specific activity comparable to that observed with phase-I (PB/beta NF) induced S9. In some instances, covalent binding was even more elevated using the BHA-induced systems as compared with traditional S9 fractions. For example, cytosol derived from BHA-administered mice was able to induce a significant binding to calf thymus DNA up to 26.2-fold increase for [14C]-1,4-dichlorobenzene, while cytosol from PB/beta NF was not. A high mutagenic response on diploid D7 strain of Saccharomyces cerevisiae as exemplified by a marked induction of mitotic gene conversion and point (reverse) mutation confirmed that BHA-derived S9 fractions activate precarcinogens to final genotoxins. Because a number of chemicals are activated by either oxidative or post-oxidative enzymes, the use of metabolizing biosystems, with an enhanced phase-II pathway, together with classical S9 fractions, can improve the sensitivity of the assay in detecting unknown genotoxins.

Biochemical and genetic interactions of two commercial pesticides with the monooxygenase system and chlorophyllin.

Two commercial preparations of atrazine and zineb were tested on a diploid D7 strain of the yeast Saccharomyces cerevisiae using cells from logarithmic growth phase (with a high level of cytochrome P-450) and from stationary growth phase. The compounds induced marked increases of both gene conversion and point mutation frequencies in the logarithmic phase cells, while in the stationary phase no genotoxic effect was observed. The results obtained employing TA98 and TA100 strains of Salmonella typhimurium (Ames test) confirmed that neither atrazine nor zineb were mutagenic. The interaction between zineb and chlorophyllin, a known antimutagen in several biological systems, has been evaluated in yeast cells from logarithmic growth phase. The results showed that chlorophyllin seems to have a protective role against the genotoxic effects of zineb. The in vivo effects on cytochrome P-450 content (Cyt. P-450) and on monooxygenase activities were examined in hepatic microsomes of induced animals (rat, pig, and rabbit) 24 hrs after acute treatment. The results obtained with atrazine showed that it caused different effects in the three animal species. Preliminary data with zineb indicated that it can act both as an inducer or as an inhibitor of the monooxygenase system, depending on the dose used.

Antimutagenesis and anticancer effects.

Cancer development is a multistage process wherein mutational events play an important role. Various antimutagen components, in particular magnesium and selenium, have been reported to have anticarcinogenic properties. These two oligoelements display different behaviors according to their concentrations. The different effects of magnesium and selenium depend on the different mechanisms of cell absorption. The importance of antimutagenesis studies in cancer prevention is reported and a review of the basic mechanisms operative in antimutagenesis, including some data on known antimutagens, is made.

Magnesium: its role in cellular functions--a review.

Magnesium has been identified as an important element in cellular functions, and its abnormalities have far-reaching effects in pathology. We reviewed its effects in the cell cycle, its physiological role, nutritional aspects, toxicity, mutagenesis, and tumorigenesis.

Effect of magnetic fields on rodent monooxygenase enzymes.

The effects of 50 Hz, 1.2 mT magnetic fields (MFs) were tested on hepatic monooxygenase enzymes of basal and beta-naphthoflavone-phenobarbital-preinduced rats and mice. An inductive effect on cytochrome P-450 level and on some enzymatic cytochrome P-450-dependent activities was observed in basal mice after MF exposure. Enzymatic activities in preinduced mice and rats were reduced by MFs, the degree of reduction depending on the enzyme. A specific inhibitory effect was determined in some of the assayed activities and in the relative peculiar P-450 isoforms detected by Western blot analysis.

Antimutagenicity in yeast.

In recent years there has been increasing interest in antimutagenesis, and studies have been done using both prokaryotic and eukaryotic systems. In eukaryotic systems the first studies were performed with different strains of Schizosaccharomyces pombe. In particular, caffeine and L-methionine were investigated. Different strains of Saccharomyces cerevisiae were employed in studies of a wide variety of compounds, including acridine, saccharin, salts, tumor promoters and co-carcinogens. Strain D7 was widely employed and antimutagenic activity of spermine, chlorophyllin, cobaltous chloride and fermented milk is reported.

Influence of cinnamaldehyde on UV-induced gene conversion and point mutation in yeast: effect on protein synthesis.

The activity of cinnamaldehyde (CIN), a bioantimutagen in bacterial systems, was tested in the D7 strain of yeast Saccharomyces cerevisiae. Yeast cells were UV-irradiated and post-incubated in liquid growth medium for 2 and 4 h with different concentrations of cinnamaldehyde. During the post-incubation period, DNA-damage-specific functions may be induced. This in turn may affect the genotoxicity and in fact a weak decrease in UV-induced convertant and revertant frequencies was observed after 4 h of post-incubation. The presence of CIN in the growth medium increased the UV-induced gene conversion and reversion. The addition of cycloheximide abolished this effect. To evaluate the CIN effect on protein synthesis, extracts of cells UV-treated and post-incubated for 2 h in the presence of 35S-methionine were performed. SDS-gel electrophoresis demonstrated the inhibitory effect of CIN on a UV-specific protein. This work suggests that CIN might interfere with DNA-damage-inducible systems although it did not exert an antimutagenic activity in our experimental conditions.

Genotoxicity of vanadium compounds in yeast and cultured mammalian cells.

The ability of vanadium compounds to induce genetic activity was investigated in D7 and D61M strains of Saccharomyces cerevisiae and in Chinese hamster V79 cell line. In our previous work, ammonium metavanadate (pentavalent form, V5) induced mitotic gene conversion and point reverse mutation in the D7 strain of yeast. The genotoxicity was reduced by the presence of S9 fraction, which probably reduced pentavalent vanadium to the tetravalent form. In the present study, vanadyl sulfate (tetravalent form, V4) induced no convertants and revertants in yeast cells harvested from stationary growth phase. With yeast cells from logarithmic growth phase, which contain high levels of cytochrome P-450, a significant increase in genetic effects was observed. Further experiments, performed by treating cells harvested from logarithmic growth phase in the presence of cytochrome P-450 inhibitors, indicated that the monooxygenase system influenced the genotoxicity of metavanadate while the genetic activity of vanadyl remained unaffected. Aneuploidy effect in the D61M strain of Saccharomyces cerevisiae was induced by either V5 or V4, confirming that vanadium compounds are potentially antitubulin agents in eukaryotic cells. Although these compounds are very toxic in V79 cells, no mutagenic effect was observed in the presence or in the absence of S9 fraction.

Vanadium: genetical and biochemical investigations.

Ammonium metavanadate was studied for its ability to induce mitotic gene conversion and reverse point mutation in the D7 strain of Saccharomyces cerevisiae. Metavanadate increased the convertant and revertant frequencies; the highest activity was observed without metabolic activation. This indicated that the S9 hepatic fraction and yeast cells in logarithmic phase (and containing a high level of cytochrome P450) biotransform vanadate, probably reducing it to vanadyl. In addition, the effect of ammonium metavanadate on the hepatic monooxygenase system was studied in mice by measuring the level of cytochrome P450 and determining the activities of aminopyrine N-demethylase, p-nitroanisole O-demethylase and 7-ethoxycoumarin O-deethylase in mouse liver microsomal fraction. The results indicated that this compound reduced mono-oxygenase activity and also the level of cytochrome P450.