Alteration of xenobiotic metabolizing enzymes by resveratrol in liver and lung of CD1 mice.

Conflicting data on the anticancer properties of the polyphenolic natural product resveratrol (RSV) have been reported. Since the inhibition of "bioactivating" Phase-I xenobiotic metabolizing enzymes (XMEs) and/or induction of "detoxifying" Phase-II XMEs have long been considered important cancer chemopreventive strategies, in the current study we investigated the effect of RSV treatment on several Cytochrome P450 (CYP)-dependent oxidations and Phase-II markers in liver and lung subcellular preparations from CD1 male mice. These mice were i.p treated with RSV (25 or 50mg/Kg b.w.) daily for one or for seven consecutive days. Using either specific probes for different CYPs, or the regio- and stereo-selective metabolism of testosterone, we found that most of the Phase-I XMEs were significantly suppressed (up to approximately 61% loss for the CYP3A1/2-linked 6 beta-hydroxylation of testosterone in liver and up to approximately 97% loss for 2 alpha-hydroxylase in lung) following RSV treatment for 7 days at 50mg/kg b.w. Glutathione S-transferase was significantly inhibited, particularly in lung (approximately 76% loss of activity) after single administration of 25mg/kg b.w. A different response for the UDP-glucuronosyl transferase was observed, where a significant induction was seen (approximately 83%) in the liver and a significant reduction was observed in the lung (up to approximately 83% loss) following treatment with 25mg/kg b.w. for seven days. These data indicate that murine XMEs are altered by RSV, and that this alteration is dependent on the RSV dose, duration and way of administration. These results could provide mechanistic explanations for the conflicting chemopreventive results reported for RSV.

A test of the nature of cosmic acceleration using galaxy redshift distortions.

Observations of distant supernovae indicate that the Universe is now in a phase of accelerated expansion the physical cause of which is a mystery. Formally, this requires the inclusion of a term acting as a negative pressure in the equations of cosmic expansion, accounting for about 75 per cent of the total energy density in the Universe. The simplest option for this 'dark energy' corresponds to a 'cosmological constant', perhaps related to the quantum vacuum energy. Physically viable alternatives invoke either the presence of a scalar field with an evolving equation of state, or extensions of general relativity involving higher-order curvature terms or extra dimensions. Although they produce similar expansion rates, different models predict measurable differences in the growth rate of large-scale structure with cosmic time. A fingerprint of this growth is provided by coherent galaxy motions, which introduce a radial anisotropy in the clustering pattern reconstructed by galaxy redshift surveys. Here we report a measurement of this effect at a redshift of 0.8. Using a new survey of more than 10,000 faint galaxies, we measure the anisotropy parameter beta = 0.70 +/- 0.26, which corresponds to a growth rate of structure at that time of f = 0.91 +/- 0.36. This is consistent with the standard cosmological-constant model with low matter density and flat geometry, although the error bars are still too large to distinguish among alternative origins for the accelerated expansion. The correct origin could be determined with a further factor-of-ten increase in the sampled volume at similar redshift.

Perturbation of murine liver cyp-superfamily of isoforms by different combinations of pesticide mixtures.

It was previously found that fenarimol, vinclozolin or acephate, three of the most used pesticides worldwide, provoked a marked perturbation of murine cytochrome P450 (CYP)-linked monooxygenases. Here, to more closely mimic human exposure, it was investigated whether different pesticide combinations administered i.p. in male Swiss Albino CD1 mice in single or repeated fashion (daily, for three consecutive days), affect CYP-dependent oxidations. The four simulated mixtures showed a complex pattern of CYP induction and suppression, especially after repeated injection. For example, while fenarimol alone was the most inducing agent--reaching a 79-fold increase over control in testosterone 2alpha-hydroxylase--followed by vinclozolin and acephate, coadministration with the former markedly reduced induction. Coadministration with vinclozolin, determined various positive and negative modulations. An increase of CYP2B1/2 and CYP3A1/2-associated oxidases and a decrease of ethoxycoumarin metabolism was observed in the acephate and vinclozolin mixture. An equivalent or reduced CYP expression, if compared to double combinations, was seen using the complete mixture. Taken as a whole, the unpredictability of the recorded effects with simple mixtures, shrinks the misleading extrapolation performed on a single pesticide. If reproduced in human, such changes, altering either endogenous metabolism or biotransformation of ubiquitous toxins, might have public health implications.

Perturbation of rat hepatic metabolising enzymes by folic acid supplementation.

An adequate folate intake minimizes the risk of various cancers and other disorders such as vascular diseases and neural tube defects. However, meta-analyses revealed difficulties in supporting the relationship between folate intake and the risk of cancer. Interestingly, there have been no reports to date on the potential ability of folate to modulate xenobiotic metabolising enzymes (XMEs), the inhibition of bioactivating Phase-I XMEs and/or induction of detoxifying Phase-II XMEs being one of the most evoked cancer chemopreventive strategies. Here, several CYP-dependent oxidations were studied in liver sub-cellular preparations from Sprague-Dawley rats receiving rodent chow supplemented with folic acid daily, for 1 or 2 consecutive months. Using either specific substrates as probes of different CYP isoforms or the regio- and stereo-selective metabolism of testosterone as a multibiomarker, we found that folic acid markedly inactivated most of the Phase-I XME analysed; up to 54% for the CYP1A1-linked deethylation of ethoxyresorufin in males, and up to 86% for the testosterone 2alpha-hydroxylase (CYP2C11) in females, after 2 months treatment. The Phase-II marker glutathione S-transferase significantly increased (~107%) after 1 month of supplementation in females only. These changes, if reproduced in humans might have public health implications. These data suggest caution in performing folate chemoprevention trials before its overall toxicological characterization has been fully addressed.

A large population of galaxies 9 to 12 billion years back in the history of the Universe.

To understand the evolution of galaxies, we need to know as accurately as possible how many galaxies were present in the Universe at different epochs. Galaxies in the young Universe have hitherto mainly been identified using their expected optical colours, but this leaves open the possibility that a significant population remains undetected because their colours are the result of a complex mix of stars, gas, dust or active galactic nuclei. Here we report the results of a flux-limited I-band survey of galaxies at look-back times of 9 to 12 billion years. We find 970 galaxies with spectroscopic redshifts between 1.4 and 5. This population is 1.6 to 6.2 times larger than previous estimates, with the difference increasing towards brighter magnitudes. Strong ultraviolet continua (in the rest frame of the galaxies) indicate vigorous star formation rates of more than 10-100 solar masses per year. As a consequence, the cosmic star formation rate representing the volume-averaged production of stars is higher than previously measured at redshifts of 3 to 4.

CYP superfamily perturbation by diflubenzuron or acephate in different tissues of CD1 mice.

This work aimed to investigate whether the insecticide acephate (125 or 250 mg/kg b.w.) or diflubenzuron (752 or 1075 mg/kg b.w.), two of the most widely used pesticides worldwide, impairs CYP-linked murine metabolism in liver, kidney and lung microsomes after repeated (daily, for three consecutive days) i.p. administration. The regio- and stereo-selective hydroxylation of testosterone was used as multibiomarker of different CYP isoforms. Both gender and tissue specific effects were observed. Lung was the most responsive tissue to induction by lower diflubenzuron dose, as exemplified by the marked increase of testosterone 7alpha-hydroxylation (CYP2A) (up to 13-fold) in males. Higher dose produced a generalized inactivation. At the lower dose acephate induced 6beta- (CYP3A1/2, liver) as well as 2beta- (CYP2B1/2, kidney) hydroxylase activities ( approximately 5 and approximately 4-fold increase, respectively) in males. In females, a marked suppression of the various hydroxylations was observed. At 250 mg/kg of acephate, animals did not survive. Induction of the most affected isoforms was sustained by immunoblotting analysis. Corresponding human CYP modulations might disrupt normal physiological functions related to these enzymes. Furthermore, the co-mutagenic and promoting potential of these pesticides, phenomena linked to CYP upregulation (e.g. increased bioactivation of ubiquitous pollutants and generation of oxygen free radicals) are of concern for a more complete definition of their overall toxicological potential.

Old galaxies in the young Universe.

More than half of all stars in the local Universe are found in massive spheroidal galaxies, which are characterized by old stellar populations with little or no current star formation. In present models, such galaxies appear rather late in the history of the Universe as the culmination of a hierarchical merging process, in which larger galaxies are assembled through mergers of smaller precursor galaxies. But observations have not yet established how, or even when, the massive spheroidals formed, nor if their seemingly sudden appearance when the Universe was about half its present age (at redshift z approximately 1) results from a real evolutionary effect (such as a peak of mergers) or from the observational difficulty of identifying them at earlier epochs. Here we report the spectroscopic and morphological identification of four old, fully assembled, massive (10(11) solar masses) spheroidal galaxies at l.6 < z < 1.9, the most distant such objects currently known. The existence of such systems when the Universe was only about one-quarter of its present age shows that the build-up of massive early-type galaxies was much faster in the early Universe than has been expected from theoretical simulations.

Effects of trivalent chromium on hepatic CYP-linked monooxygenases in laying hens.

Chromium is an essential nutrient required for the metabolism of carbohydrates and lipids in humans and many animal species. We evaluated whether feeding laying hens with high levels of different chemical forms of trivalent chromium may affect hepatic metabolizing cytochrome P-450 (CYP)-linked enzymes. Modulation of CYP-dependent monooxygenases (which play a pivotal role in the endogenous metabolism) by Cr(III) was tested using either specific substrates as probes of different CYPs or testosterone as a multi-bioprobe. The CYP-supported oxidases were studied in liver microsomes from laying hens fed with diet supplemented with either 25 or 50 ppm chromium chloride as a yeast or as aminoniacinate. Although at 25 ppm no appreciable effects were observed, at 50 ppm a general inactivation of the recorded monooxygenases (ranging from 34% loss for ethoxycoumarin O-deethylase with chromium chloride to 85% loss for O-deethylation of ethoxyresorufin with either chromium yeast or aminoniacinate) were achieved in the supplemented groups vs controls. The only exception was the O-dealkylation of pentoxyresorufin, which was significantly boosted by both chromium yeast (up to 65% increase) and chromium aminoniacinate (up to 141%). Measurements of the regio- and stereoselective hydroxylation of testosterone used as a multi-bioprobe corroborated the inactivating properties of Cr(III) at the higher dose. Taken as a whole, these findings seem to indicate that high levels of Cr(III) supplementation can substantially impair CYP-catalysed drug metabolism in laying hens.

Induction of cytochrome P450 enzymes and over-generation of oxygen radicals in beta-carotene supplemented rats.

Effects of beta-carotene (betaCT) on microsomal CYP-linked monooxygenases were investigated using both the regio- and stereo-selective hydroxylation of testosterone (as multibiomarker) and highly specific substrates as probes of various isoenzymes. CYP-catalyzed reactions were studied in the liver, kidney, lung and intestine of Sprague-Dawley rats of both sexes supplemented with 250 or 500 mg/kg body wt betaCT (per os) in a single or repeated (daily for 5 days) fashion. Generalized boosting effects (2-15-fold increases) were observed in the various tissues for carcinogen metabolizing enzymes associated with CYP1A1/2, CYP3A1/2, CYP2E1, CYP2B1/2 and CYP2C11. Induction of the most affected CYPs was corroborated by western blot linked to densitometric analyses. Measurement of reactive oxygen species (ROS) produced by subcellular preparations from either control or betaCT supplemented rats was performed by EPR detection of the nitroxide radical yielded by the reaction with ROS of the hydroxylamine spin probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate. Marked ROS over-generation associated with CYP induction (up to 33-fold increase in the liver) was recorded in the various organs (liver > lung > intestine > kidney). CYP and ROS induction are substantially in keeping with the concentration of betaCT accumulated in the various tissues, the liver being the most affected organ. These findings are consistent with the concept that betaCT is a pro-oxidant and potentially co-carcinogenic pro-vitamin, and may help explain why, in large quantities, it can have harmful effects in humans.

Mechanism for the prevention of cholestasis involving cytochrome P4503A overexpression.

BACKGROUND: To clarify the preventive effect of taurohyodeoxycholic acid on liver cholestasis induced by toxic bile acids in rats, we evaluated whether modulation of cytochrome P4503A-linked oxidases is involved in the hepatic bile acid retention and secretion mechanism. We investigated whether the safe or the toxic taurochenodeoxycholic acid, administered singly or together, affects cytochrome P450-catalyzed drug metabolism or biliary parameters. We also considered whether the inhibition of the P-glycoprotein export pump by vinblastine might be related to cytochrome P4503A overexpression. METHODS: Hydroxylation of testosterone and N-demethylation of aminopyrine were studied in subcellular rat liver preparations after intravenous infusion of hepatoprotective and toxic bile acids administered singly or together. Bile flow, calcium secretion, biliary enzymes activity, and secretion rates of the endogenous and administrated bile acids were determined. CYP3A-dependent monooxygenases were also measured in the same coinfusion model in the presence of vinblastine. RESULTS: Although wide modulation of the activities of different P450 subfamily of isoenzymes was seen, P4503A-associated monooxygenases showed similar patterns in the various situations, i.e., induction by taurohyodeoxycholic acid, reduction by taurochenodeoxycholic acid, and protection (intermediate induction) in the coinfusion experiments. This correlates well with biliary parameters demonstrating the hepatoprotective ability of taurohyodeoxycholic acid. Coadministration of bile acids and vinblastine significantly modifies CYP3A-linked activities. CONCLUSIONS: Bile acid structure seems to be linked with hepatotoxicity/hepatoprotection and P4503A modulation. Taurohyodeoxycholic acid could be therapeutic in cholestatic liver disease by inducing P4503A; we can hypothesize that an associated P-glycoprotein expression might facilitate biliary excretion of toxic taurochenodeoxycholic acid accumulated in the liver during cholestasis.

Effect of liquorice and glycyrrhizin on rat liver carcinogen metabolizing enzymes.

We investigated the effect of single or repeated intake of conspicuous amounts of licorice root extract (LE, 3138 or 6276 mg/kg body weight (bw) per os) or its natural constituent glycyrrhizin (G, 240 or 480 mg/kg bw per os) on Sprague-Dawley rat liver monooxygenases. Whereas a single LE or G dose was unable to affect CYP superfamily, four daily doses induced CYP3A, CYP1A2 and to varying extents CYP2B1-linked monooxygenases. A boosting effect on testosterone 6beta- (CYP3A1/2, CYP1A1/2), 7alpha- (CYP1A1/2, CYP2A1), 16alpha- (CYP2B1, CYP2C11), 2alpha- (CYP2C11) and 2beta- (CYP3A1, CYP1A1) -dependent oxidases as well as on androst-4-ene-3,17-dione- (CYP3A1/2) -supported monooxygenases were also achieved. Harmful outcomes associated to CYP changes (e.g. cotoxicity, cocarcinogenicity and promotion) may be of concern.

Bile acid structure and selective modulation of murine hepatic cytochrome P450-linked enzymes.

We examined the effects of the administration of different bile acids on in vivo hepatic murine cytochrome P450 (CYP) content, nicotinamide adenine dinucleotide phosphate (NADPH)-CYP-reductase, and individual mixed-function oxidases (MFOs). Neither CYP level nor reductase were appreciably affected by single intraperitoneal administration of taurodeoxycholic acid (TDCA) (12.2 or 24.4 mg x kg(-1) bw). MFO to various isoenzymes were slightly reduced 24 hours after treatment. Taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) both induced CYP, reductase, and MFOs. CYP3A1/2-linked activity (i.e., testosterone 6beta-hydroxylase, and N-demethylation of aminopyrine) in a dose-dependent fashion was enhanced ( approximately 2-3-fold). CYP2E1- (hydroxylation of p-nitrophenol), CYP1A2-(O-demethylation of methoxyresorufin), CYP2A1/2- and CYP2B1/2-(6alpha-hydroxylase), and CYP2B9- (16alpha-hydroxylase) dependent MFOs, as well as 7alpha-, 16beta-, 2alpha-, and 2beta-hydroxylations, were all significantly induced by THDCA. Apart from alkoxyresorufin metabolism and a modest CYP2E1 increase, TUDCA behaved like THDCA. A generalized induction was also recorded after ursodeoxycholic acid (UDCA) administration. THDCA and TDCA did not show substantial differences in the N-demethylation of aminopyrine when different species (rat vs. mouse) and administration route (intraperitoneal vs. intravenous) were compared. Results on the most affected isoenzymes, CYP3A1/2 (THDCA, TUDCA, and UDCA) and CYP2E1 (UDCA), were sustained by means of Western immunoblotting. CYP3A induction was paralleled by a corresponding increase in mRNA. These data could partially explain the therapeutic mechanism of UDCA, TUDCA, and THDCA in chronic cholestatic liver disease. CYP3A induction, which is linked to P-glycoprotein (Pgp) family overexpression, may enhance hepatic metabolism, transport, and excretion of toxic endogenous lipophilic bile acids.

Induction and suppression of murine CYP-mediated biotransformation by dithianon: organ- and sex-related differences.

With the aim of evaluating the co-carcinogenic properties of dithianon, the regio- and stereo-selective hydroxylation of testosterone was used as a multibiomarker of effect for cytochrome P450 (CYP) changes. CYP-catalysed reactions have been studied in liver, kidney and lung microsomes from male and female Swiss albino CD1 mice treated i.p. with single (3 or 6 mg/kg body wt.) or repeated (3 mg/kg body wt. daily for 3 days) doses of this fungicide. Induction or suppression was recorded under various situations in different organs and sexes. In liver, all testosterone hydroxylase (TH) activities were increased in the single treatment from 2.8- (6beta-, 16alpha- and 16beta-TH activities) to 16-fold (2beta-TH activity) in males at the lower dose. In contrast, activities were reduced from 33.3% (16beta- and 17-TH activities, lower dose) to 66.4% (16beta-TH activity, higher dose) in females. In kidney, a similar pattern of modulation was achieved: induction from 2.9- to 5-fold (6beta- and 2alpha-TH activities, higher and lower doses, respectively) in males; suppression from 47.4 to 50.2% (2alpha- and 2beta-TH activities, either at lower or higher doses) in females. In lung, a significant induction ranging from 7.1- to 29.3-fold (16alpha- and 2alpha-TH activities, respectively, lower dose) in males, and up to a 7-fold increase (2beta-TH activity, higher dose) in females was obtained. After repeated treatment, hepatic 6beta-, 16beta-, 2alpha- and 2beta-TH activities were reduced up to approximately 60% in males, whereas no effect was seen in females. In extrahepatic tissues, a generalized increase of different THs was observed. The increase of 6beta-TH activity (CYP3A-linked), one of the most representative isoforms in humans, was sustained in liver and kidney by means of Western immunoblotting, using rabbit polyclonal antibodies anti CYP3A1/2. On the whole, a complex pattern of induction/suppression of CYP-dependent reactions was achieved depending on sex and tissue. The data are consistent with co-toxic, co-carcinogenic and promoting potentials of this fungicide and provide information of interest in evaluating the risk associated with human exposure.

The nature of prooxidant activity of vitamin C.

It was recently reported that vitamin C (500 mg/day for 6 weeks) administered as a dietary supplement to healthy humans exhibits a prooxidant, as well as an antioxidant effect in vivo. Here we show that high intakes of vitamin C (500 mg/kg b.w. for 4 days) in the rat are able to markedly induce hepatic cytochrome P4502E1-linked monooxygenases, measured as p-nitrophenol hydroxylase activity and corroborated by means of Western blot analyses. Furthermore, using Electron Paramagnetic Resonance Spectroscopy (EPR) coupled to a spin-trapping technique, we have also found that this induction generates large amounts of the anion radical superoxide (O2-). Therefore we can conclude that the adverse prooxidant outcomes (i.e. oxidative DNA damage) associated to vitamin C supplementation, being associated to a typical reversible boosting effect (i.e. enzymatic induction), may be easily controlled by a discontinuous supply. However, since the induced P4502E1 isoforms by vitamin C are responsible for ethanol metabolism to highly reactive radicals, care should be taken even in moderate drinkers.

Molecular non-genetic biomarkers of effect related to methyl thiophanate cocarcinogenesis: organ- and sex-specific cytochrome P450 induction in the rat.

We used selective biochemical markers of effect to evaluate some non-genotoxic cocarcinogenic properties of methyl thiophanate (MTH) associated with cytochrome P450 (CYP) changes. Several CYP-dependent reactions were monitored in the liver, kidney and lung microsomes of male and female Sprague-Dawley rats treated (i.p.) with a single (285 or 570 mg/kg body weight) or repeated (daily 285 or 570 mg/kg body weight for three consecutive days) doses of this pesticide. No significant changes in absolute or relative liver, kidney and lung weights were observed after MTH injection. Highly specific substrates were used as probes of different isoforms, such as CYP1A1, 1A2, 2B1, 2E1 and 3A. A complex pattern of CYP induction, including organ- and sex-related differences, was observed, particularly in the liver (CYP3A, 2B1), kidney (CYP1A1, 2E1) and lung (CYP3A, 1A1). In the liver, an increase up to 29-fold in the 2B1-like activity, probed by the O-dealkylation of pentoxyresorufin, was observed at lower dose in both sexes, and the induction of CYP 1A2-mediated methoxyresorufin O-demethylase activity (up to 3.6-fold) was recorded at the higher dose in males. In the kidney, the O-deethylation of ethoxyresorufin (CYP1A1-linked) was increased up to 28.2-fold and the CYP2E1-dependent p-nitrophenol hydroxylases were enhanced up to 6.3-fold in females receiving higher multiple MTH administration. In the lung, the CYP3A-associated activity was the most induced oxidases, as exemplified by the marked increase in the O-demethylation of aminopyrine (up to 3.6-fold) in males. A weak, although significant, reduction of CYP2B1-linked oxidases was also observed in repeated treatment in the kidney (males) and lung (females). These results suggest that the induction of CYP-catalyzed drug metabolism by prolonged exposure to MTH may result in accelerated metabolism of coadministered drugs with important implications for their disposition Together with an alteration of endogenous metabolism, the adverse effects associated with CYP changes such as toxicity/cotoxicity, cocarcinogenicity and promotion may also have clinical consequences.

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.

Cancer chemoprevention: some complications and limitations.

Chemopreventive strategies are very attractive and have earned serious consideration as a potential means of controlling cancer incidence. However, the use of some anti-initiating entities (enzyme inducers or inhibitors) devised to reduce tumor initiation is controversial. Indeed, considering the double-edged-sword (activating or detoxifying) nature of drug metabolizing enzymes, any attempt to modulate such catalysts by dietary components (including drugs) may lead to cancer risk.

Effect of licorice and glycyrrhizin on murine liver CYP-dependent monooxygenases.

This study is aimed to investigate the effect of the prolonged intake of conspicuous amounts of licorice (LE), or its natural constituent glycyrrhizin (G) on murine liver CYP-catalyzed drug metabolism. For this purpose the modulation of the regio- and stereo-selective hydroxylation of testosterone, together with the use of highly specific substrates as probes for different CYP isoforms such as ethoxyresorufin (CYP1A1), methoxyresorufin (1A2), pentoxyresorufin (2B1), p-nitrophenol (2E1) and aminopyrine (3A), were investigated. Daily doses of licorice root extract (3,138 or 6,276 mg/kg b.w. per os), or G (240 or 480 mg/kg b.w. per os), were administered to different groups of Swiss Albino CD1 mice of both sexes for 1, 4 or 10 consecutive days. While a single LE or G dose was unable to affect the multienzymatic CYP-system, using both schedules of repeated treatment, either LE or G were able to significantly induce hepatic CYP3A- and, to a lesser extent, 2B1- and 1A2-dependent microsomal monooxygenase activities, as well as 6beta- (mainly associated to CYP3A), 2alpha-, 6alpha- (CYP2A1, 2B1), 7alpha-, 16alpha- (CYP2B9) and 16beta-testosterone hydroxylase (TH) activities in male and female mice. Data on CYP3A modulation, the major isoform present in human liver, was confirmed by using Western immunoblotting with anti-CYP3A1/2 rabbit polyclonal antibodies raised against purified rat CYP3A. Northern blotting analysis using CYP3A cDNA biotinylated probe showed that the expression of such isozyme is regulated at the mRNA level. These results suggest that the induction of cytochrome P450-dependent activities by the prolonged intake of high LE or G doses, may result in accelerated metabolism of coadministered drugs with important implications for their disposition. The adverse effects associated with CYP changes such as toxicity/cotoxicity and comutagenicity may also have clinical consequences.

Development of basal and induced testosterone hydroxylase activity in the chicken embryo in ovo.

1. The sensitivity of the developing embryo to xenobiotics is highly dependent on the expression of metabolizing enzymes including cytochromes P450 (CYP). In the present study, therefore, the ontogeny of the CYP-dependent system in the chick was investigated with testosterone hydroxylase activity as a marker of CYP expression. 2. Chicken embryo livers were assayed for basal and phenobarbitone (PB)-induced regio- and stereo-selective testosterone hydroxylase activity, from the first appearance of the liver as a discrete organ at 5 days of incubation through day 10 posthatching. In addition, whole embryo preparations were assayed at 3 and 4 days of incubation. 3. Whereas testosterone 16 beta-hydroxylase and androst-4-ene-3, 17-dione-linked activities were expressed during all stages of embryonic development, testosterone 6 alpha-, 6 beta-, 7 alpha- and 16 alpha-hydroxylase activities were observed only in basal embryos from 8 days of incubation. Furthermore, testosterone 2 alpha- and 2 beta-hydroxylase activities were detected exclusively from 10 days of incubation onward. All activities increased steadily throughout development as did the responsiveness of the embryonic liver to PB induction. 4. A typical pattern of development with a higher activity from 10 to 14 days of incubation (testosterone 16 alpha-, 7 alpha-, 6 alpha- and 2 beta-hydroxylase activities; up to 4.1 +/- 0.3 pmol mg-1 protein min-1 at 13 days of incubation for testosterone 7 alpha-hydroxylase) or shifted to 14 to 18 days of incubation (testosterone 6 beta-, 2 alpha- and 16 beta-hydroxylase activities: up to 56.6 +/- 1.4 pmol mg-1 protein min-1 at 16 days of incubation for testosterone 6 beta-hydroxylase) was observed. There was a tendency towards an increased activity for all activities around hatching, specifically from 19 days of incubation to 4 days posthatching (up to 1,759.3 +/- 179.4 pmol mg-1 protein min-1 at 1 day posthatching for androst-4-ene-3,17-dione-linked activity). 5. The highest level of PB-induced enzyme activity was observed for testosterone 2 alpha-hydroxylase activity (95.14 +/- 7.35 and 660.19 +/- 45.27 pmol mg-1 protein min-1) at 12 days of incubation and day 3 posthatching, respectively. Except for testosterone 2 alpha- and 2 beta-hydroxylase activities at 3 to 4 days of incubation, all metabolites were detectable during the first period of organogenesis in the presence of PB. 6. The use of highly specific substrates, studies on the immunoinhibition of metabolism by polyclonal antibodies raised against highly purified rat CYPs, and the use of selective inhibitors seemed to reveal a wide pleiotropic response with the possible presence in liver of PB-treated chickens of CYP1A together with CYP2HI/H2, CYP2E and CYP3A.

Molecular non-genetic biomarkers of effect related to acephate cocarcinogenesis: sex- and tissue-dependent induction or suppression of murine CYPs.

The aim of this work was to study the ability of the organophosphate insecticide acephate to alter some biochemical markers of effect related non-genetic cocarcinogenesis. For this purpose, selective CYP-dependent reactions have been examined in liver, kidney and lung microsomes of male and female Swiss albino CD1-mice treated (i.p.) with a 125 or 250 mg/kg b.w. dose of this pesticide. High specific substrates were used as a probe of various isozymes, such as CYP 1A1, 1A2, 2B1, 2E1 and 3A. Maked organ- and sex-related differences in either inducive or suppressive response by acephate depict a complex pattern of CYP modulation with the kidney being more responsive to 3A induction (up to 6.9-fold increase, male) and the lung to 2B1 suppression (up to 70% loss, mainly female). In the liver, a 2.7-fold increase in the 3A-like activity, probed by the O-demethylation of aminopyrine, in the O-deethylation of phenacetin (1.8-fold increase, 1A2), as well as in the hydroxylation of p-nitrophenol (1.6-fold increase, 2E1) was observed in male animals at a lower dose. In contrast, a marked reduction of CYP 1A1-mediated ethoxyresorfin O-deethylase activity ranging from 43% (lower dose) to 44% loss (higher dose) in female and male mice, respectively, and of CYP 2B1-mediated pentoxyresorufin O-dealkylase (3% loss, female) was achieved. In the kidney, an increase in the 'mixed' ethoxycoumarin O-deethylase (up to 2-fold) as well as in the 2B1-like activity (up to 2.8-fold) was also recorded in males at 250 mg/kg. Once again, in the lung, a different behaviour on 3A isoforms between female (approximately 2-fold increase) and male (44% loss) was seen at a lower dose. The specificity of CYP changes was corroborated by means of Western immunoblotting analysis using rabbit polyclonal antibodies, anti-CYP 3A1/2 and 2E1. Taken together, these data indicate a possible toxic/cotoxic, cocarcinogenic and promoting potential of acephate.