Protective effects of garlic sulfur compounds against DNA damage induced by direct- and indirect-acting genotoxic agents in HepG2 cells.

The aim of this study was to assess the antigenotoxic activity of several garlic organosulfur compounds (OSC) in the human hepatoma cell line HepG2, using comet assay. The OSC selected were allicin (DADSO), diallyl sulfide (DAS), diallyl disulfide (DADS), S-allyl cysteine (SAC) and allyl mercaptan (AM). To explore their potential mechanisms of action, two approaches were performed: (i) a pre-treatment protocol which allowed study of the possible modulation of drug metabolism enzymes by OSC before treatment of the cells with the genotoxic agent; (ii) a co-treatment protocol by which the ability of OSC to scavenge direct-acting compounds was assessed. Preliminary studies showed that, over the concentration range tested (5-100 microM), the studied OSC neither affected cell viability nor induced DNA damage by themselves. In the pre-treatment protocol, aflatoxin B1 genotoxicity was significantly reduced by all the OSC tested except AM. DADS was the most efficient OSC in reducing benzo(a)pyrene genotoxicity. SAC and AM significantly decreased DNA breaks in HepG2 cells treated with dimethylnitrosamine. Additionally, all the OSC studied were shown to decrease the genotoxicity of the direct-acting compounds, hydrogen peroxide and methyl methanesulfonate. This study demonstrated that garlic OSC displayed antigenotoxic activity in human metabolically competent cells.

Analytical method for appreciation of garlic therapeutic potential and for validation of a new formulation.

The consumption of garlic reduces the risk of cardiovascular disease and cancer, S-allylcysteine sulfoxide (alliin), allicin (DATi), diallyl disulfide (DADS), S-allylcysteine (SAC) and several storage dipeptides are the organo-sulphur compounds (OSC) involved in the protective mechanism of garlic against cardiovascular disorders and carcinogenesis. Thus it is very interesting to quantify simultaneously all these compounds in different garlic powders obtained in several cultural conditions. The quantification of OSC by a new ion-pair HPLC method allowed showing the general sulphur-dependence positive effect of garlic on cardiovascular disorder and carcinogenesis and the variable specific activity of each implicated OSC. The screening of 11 garlic tablets proposed on the market showed the variability and particularly the differential instability of each OSC. From these results, a new garlic tablet was realised and each step was controlled by this method. This analytical method proved to be a very powerful tool for the understanding of the garlic protective mechanism against cancer and cardiovascular diseases and the development and quality control of garlic tablets.

Modification of hepatic drug-metabolizing enzymes in rats treated with alkyl sulfides.

Natural compounds which elevate detoxification enzymes and/or reduce activating enzymes could be considered as good candidates to protect against cancer. In this work, we studied the modulation of hepatic drug-metabolizing enzymes in rats treated with dimethyl sulfide (DMS), dimethyl disulfide (DMDS), methylpropyl disulfide (MPDS), dipropyl sulfide (DPS), dipropyl disulfide (DPDS) and diallyl disulfide (DADS) issued from Allium species. Compounds containing methyl groups had little or no effect. Compounds with two propyl groups or two allyl groups provoked a pleiotropic response on drug-metabolizing enzymes. DPS, DPDS and DADS induced ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase and mostly pentoxyresorufin O-depentylase and decreased nitrosodimethylamine N-demethylase and erythromycin N-demethylase. These modifications of enzyme activities were accompanied by an increase of CYP 2B1,2 and a decrease of CYP 2E1, evidenced by immunoblotting. The same treatments stimulated some phase II enzyme activities such as glutathione transferase and UDP-glucuronyl transferases. This pattern of induction and/or inhibition of drug metabolizing enzymes was qualitatively similar to that elicited by the enzyme inducer, phenobarbital. The magnitude of the effects produced by DPDS was smaller than those produced by DADS and DPS. Our results suggest a possible protective effect of alkyl sulfides as well as diallyl disulfide, on the first step of carcinogenesis via the modulation of enzymes involved in carcinogen metabolism.

Ah receptor-dependent CYP1A induction by two carotenoids, canthaxanthin and beta-apo-8'-carotenal, with no affinity for the TCDD binding site.

The assays of several phase I and phase II xenobiotic-metabolizing enzyme activities, as well as CYP1A immunoblot analysis, were performed in liver microsomes and cytosol of male C57BL/6 mice (Ah receptor-responsive), of male DBA/2 mice (Ah receptor-low responsive) and of female Ah receptor gene knockout mice that were fed diets containing 300 mg/kg of a nonprovitamin A carotenoid, canthaxanthin, or a provitamin A carotenoid, beta-apo-8'-carotenal for 14 days, or which were injected i.p. with 3-methylcholanthrene. Previous studies have shown that some carotenoids, such as canthaxanthin and beta-apo-8'-carotenal, are strong inducers of liver CYP1A1 and 1A2 when given to rats. In this work, only canthaxanthin induced both CYP1A1 and 1A2 in C57BL/6 mice, whereas beta-apo-8'-carotenal induced only CYP1A2 in this strain. Neither of the two carotenoids modified CYP1A1/2 protein contents or enzyme activities in Ah receptor-low responsive DBA/2 or in Ah receptor gene knockout mice. Cytosol prepared from C57BL/6 mice liver tissue was incubated with [3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence of canthaxanthin or beta-apo-8'-carotenal and analyzed by sucrose density gradient sedimentation: neither of the carotenoids, even when present in large excess, competed with TCDD for the TCDD binding site of the cytosolic Ah receptor of C57BL/6 mice. In brief, the carotenoids canthaxanthin or beta-apo-8'-carotenal induced Cyp1a genes in mice through an Ah receptor-dependent pathway, but did not bind to the Ah receptor.

Comparative effects of flavonoids and model inducers on drug-metabolizing enzymes in rat liver.

The inducing effects of some flavonoids (flavone, flavanone, tangeretin and quercetin) and model substances have been studied in rats, and the activity and the expression of drug-metabolizing enzymes have been compared in rats. The addition of flavonoids to the diet (0.3% w/w) for 2 weeks did not change the liver cytochrome P450 content nor the activities of the NADPH-cytochrome P450 and NADH-cytochrome b5 reductases, but it affected the activities of phase I and phase II enzymes. Flavone, and to a lesser extent tangeretin, increased the activities mediated by the P450 1A1,2 (EROD) and 2B1,2 (PROD) as well as the activities of p-nitrophenol UDP-glucuronyl transferase (UGT) and glutathione transferase (GST). Flavanone mainly enhanced PROD, UGT and GST, whereas quercetin did not modify any enzyme activities. None of the tested flavonoids modulated the activities catalyzed by P450 2E1, 3A and 4A. Immunoblotting studies showed that flavone and tangeretin increased the expression of cytochrome P450 1A and 2B forms, whereas flavanone only induced cytochrome P450 2B. Flavone and to a lesser extent flavanone, markedly increased the phenol-UGT protein level. Both flavone and flavanone also increased the androsterone- and testosterone-UGTs, whereas tangeretin and quercetin did not increase any UGT isoform. We concluded that the flavonoids tested specifically affected the expression of the drug-metabolizing isozymes in rat liver, their inducing properties were dependent on their chemical structures.

Inhibition of microsome-mediated binding of benzo[a]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats.

The in vitro and in vivo effects of selected natural flavonoids (flavone, flavanone, tangeretin, quercetin, chrysin) on the microsome-catalysed binding of [3H]benzo[a]pyrene to calf thymus DNA were investigated and compared with those of two synthetic flavonoids, 7,8-benzoflavone and 5,6-benzoflavone. In vitro addition of these flavonoids (0.1 mM) to an incubation system containing hepatic microsomes prepared from Aroclor 1254-pretreated rats strongly inhibited BaP-DNA adduct formation (72-89%). The incubation of BaP with hepatic microsomes prepared from animals fed 0.3% quercetin, tangeretin and 7,8-benzoflavone for 2 weeks also resulted in less effective binding of BaP metabolites to added DNA, than with microsomes from untreated rats. Other tested compounds, chrysin, flavone, flavanone and 5,6-benzoflavone showed no or little effect. The influence of flavonoid pretreatment on hepatic microsomal enzymes involved in BaP metabolism has also been examined. Aryl hydrocarbon hydroxylase activity was moderately increased (1.5-1.8-fold) in microsomes prepared from rats fed flavone, tangeretin, 7,8-benzoflavone and 5,6-benzo-flavone. Epoxide hydrolase activity was enhanced by 7,8-benzoflavone (1,6-fold), and by flavone and flavanone (5-fold). These results confirm that flavonoids, in vitro, are potent inhibitors of carcinogen-DNA binding. Oral administration of 0.3% flavonoids alters the properties of liver microsomes, resulting in the decreased ability of BaP metabolites to bind DNA.

Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation.

The effects of in vivo administration of naturally occurring organosulfur compounds (OSCs) from Allium species were studied on the activation of several mutagens. Male SPF Wistar rats were given p.o. one of either diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) or dipropyl disulfide (DPDS) during 4 consecutive days and the ability of hepatic S9 and microsomes from treated rats to activate benzo[a]pyrene (BaP), cyclophosphamide (CP), dimethylnitrosamine (DMN), N-nitrosopiperidine (N-PiP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined in the Ames test. Administration of DAS, DPS and DPDS resulted in a significant increase of the activation of BaP, CP, N-PiP and PhIP mediated by S9 and microsomes while DADS treatment only increased the mutagenicity of PhIP. In contrast, S9 from DADS-treated rats significantly inhibited the mutagenicity of N-PiP and BaP. DAS, DADS and DPS strongly inhibited DMN mutagenicity while DPDS enhanced it. To understand the mechanisms underlying these effects, the modifications of the activities of specific isozymes of CYP involved in the activation of these mutagens were studied. DAS, DPS and DPDS strongly enhanced pentoxyresorufin O-dealkylase (PROD) activity related to CYP2B and slightly increased ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities related to CYP1A family. DADS exerted the same effects than other OSCs but to a lesser extent. p-Nitrophenol hydroxylase (PNPH) activity related to CYP2E1 was inhibited by DAS and DADS, whereas DPDS significantly increased this activity. Hence, the effects of OSCs on the mutagenicity of several genotoxic compounds are mediated by modification (enhancement or inhibition) of specific CYP involved in their activation.

Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction.

In a previous study, we showed that naturally occurring organosulfur compounds (OSCs) from garlic and onion modulated the activation of carcinogen via the alteration of cytochromes P450. The present study was undertaken to determine the incidence of the in vivo induction of phase II enzymes by individual OSCs on the genotoxicity of several carcinogens. Diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS), were administered by gavage (1mmol/kg) to male SPF Wistar rats for 4 consecutive days. The effects of treatments on phase II enzymes and on the genotoxicity of carcinogens were evaluated with hepatic cytosols and microsomes from OSCs-treated rats. DADS strongly increased all the phase II enzymes activities examined, i.e. total glutathione S-transferase (GST) activity, mu GST activity, quinone reductase (QR) activity and epoxide hydrolase (EH) activity. In addition, DADS strongly increased the protein level of rGSTP1. QR activity, total and mu GST activities were also increased by DAS and DPDS whereas DPS increased only mu GST activity and QR activity. To assess the repercussions of these inductions on the genotoxicity of carcinogens, the effects of cytosols or microsomes from OSCs-treated rats on the mutagenicity of (+)-anti-7beta,8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), styrene oxide (SO) and 4-nitroquinoline 1-oxide (4-NQO) were measured in the Ames test. DADS showed a very effective antimutagenic activity against BPDE, SO and 4-NQO. DAS reduced the mutagenicity of BPDE and SO. In contrast, DPS and DPDS showed little efficient antimutagenic activity since they only reduced the mutagenicity of BPDE and 4-NQO, respectively. Interestingly, DADS appeared to be as effective as ethoxyquin, a model inducer of phase II enzymes, in both inducing phase II enzymes and inhibiting the mutagenicity of carcinogens. This study demonstrated that the antimutagenic activities of OSCs against several ultimate carcinogens were closely related to their ability to induce phase II enzymes.

Comparison of the effects of various flavonoids on ethoxycoumarin deethylase activity of rat intestinal and hepatic microsomes.

Fifteen flavonoids were examined for their effects on the activity of 7-ethoxycoumarin O-deethylase in rat hepatic and intestinal microsomes. The effect depended on both the chemical structure of the flavonoid and the origin of the microsomes. Polyhydroxylated flavonoids with a C2-C3 double bond (flavones and flavonols) were more effective inhibitors of the enzyme in both hepatic and intestinal microsomes than were the reduced homologues (flavanonols, flavanones and flavan-3-ols). In contrast, flavones lacking hydroxyl substituents (e.g. 5,6-benzoflavone, 7,8-benzoflavone and flavone) increased ethoxycoumarin deethylase activity in liver microsomes although they had an inhibitory effect in intestinal microsomes.

The influence of food flavonoids on the activity of some hepatic microsomal monooxygenases in rats.

The effects of several food flavonoids on the activities of some drug-metabolizing enzymes in rat-liver microsomes were determined in vivo. When quercetin, chrysin, hesperetin and (+)-catechin were injected ip at doses of 150 mg/kg body weight/day for 3 days, quercetin and chrysin stimulated aminopyrine demethylase. When quercetin, rutin, hesperetin and (+)-catechin were fed as components (0 . 1%) of purified diets administered to rats for 4 wk, rutin and (+)-catechin increased microsomal cytochrome b5 levels and quercetin and (+)-catechin increased aminopyrine demethylase activity. On the other hand rutin, quercetin and hesperetin inhibited NADH-cytochrome c reductase. Aniline hydroxylase was not affected by any of the flavonoids administered by either route. The results show that each flavonoid affects different components of the monooxygenase system. The fact that some flavonoids give different effects according to the route and/or the duration of administration is discussed.

Modifications of hepatic drug metabolizing enzyme activities in rats fed baobab seed oil containing cyclopropenoid fatty acids.

The effects on drug metabolizing enzymes of cyclopropenoid fatty acids present in baobab seed oil were evaluated in rats fed either a diet with baobab seed oil (1.27% cyclopropenoid fatty acids in the diet) or a diet with heated baobab seed oil (0.046% cyclopropenoid fatty acids in the diet). Comparison was made with rats fed a mixture of oils that contained no cyclopropenoid fatty acid. Rats fed baobab oil showed retarded growth. In comparison with the other groups, the relative liver weights were markedly increased whereas cytochrome P-450 content and NADPH cytochrome c reductase and NADH cytochrome c reductase activities were decreased. In rats fed the heated baobab oil the relative liver weight was decreased and the cytochrome P-450 level and reductase activities were increased relative to levels in rats fed the unheated oil. Ethoxycoumarin deethylase, ethoxyresorufin deethylase and pentoxyresorufin depentylase activities, expressed on the basis of cytochrome P-450, were greater in the group fed unheated baobab seed oil. Cytosolic glutathione transferase activity was markedly decreased in rats fed fresh baobab seed oil and heating the oil, which reduced the content of cyclopropenoid fatty acids, led to a considerable increase of this activity. UDP-glucuronyl transferase activities were not modified by the type of oil included in the diet. It is possible that the mechanisms of action of cyclopropenoid fatty acids are related to alterations of membrane lipid composition or microsomal proteins.

Comparison of the effects of feeding quercetin or flavone on hepatic and intestinal drug-metabolizing enzymes of the rat.

The influence of dietary flavone and quercetin on the components of the drug metabolizing enzyme system was examined in the liver and small intestine of the rat. Quercetin given at a concentration of 1% in the diet for 14 days produced no significant changes on phase I or phase II enzyme activities. In contrast, 0.25% flavone caused significant increases in relative liver weight, microsomal and cytoplasmic proteins, and cytochrome P-450 content. The activities of hepatic ethoxyresorufin, pentoxyresorufin and ethoxycoumarin deethylases were significantly increased (by 20, 30 and 2.5-fold, respectively) over control levels. Hepatic UDP-glucuronyl transferase and glutathione transferase activities were increased 3-4-fold. In contrast, flavone induced no changes in these two intestinal enzyme activities. It is concluded that flavone produces an induction that shows both phenobarbital- and methylcholanthrene-type characteristics and that its effects on conjugating enzymes is tissue specific.

Induction of hepatic drug-metabolizing enzymes by cyclic fatty acid monomers in the rat.

The effects of cyclic monomers on the activities of several drug-metabolizing enzymes were evaluated. Female Wistar rats were fed, for 4 wk, a semi-synthetic diet containing different quantities of cyclic monomers isolated from linseed oil heated at 275 degrees C for 12 hr under nitrogen. Microsomal proteins and cytochrome c were significantly increased in rats fed a diet containing 0.1 or 1% cyclic monomers. Aminopyrine demethylation, a model reaction preferentially induced by phenobarbital, was increased by this treatment. NADPH-cytochrome P-450 reductase was also stimulated. Moreover, ethoxyresorufin deethylation, known to be greatly increased by methylcholanthrene-type inducer was only increased threefold by this treatment. The activity of p-nitrophenol UDP-glucuronosyl transferase decreased while the conjugation of bilirubin was stimulated. These results suggest that cyclic monomers isolated from heated linseed oil show some characteristics of phenobarbital-type inducers.

Effects of beta-carotene and canthaxanthin on liver xenobiotic-metabolizing enzymes in the rat.

The activities of several phase I and phase II xenobiotic-metabolizing enzymes have been measured in liver microsomes and cytosol of male rats that had been fed for 15 days with diets containing beta-carotene or canthaxanthin (300 mg/kg diet) or an excess of vitamin A (70,000 IU/kg diet), or to which beta-carotene had been administered by ip injections (7 x 10 mg/kg body weight). Microsomal cytochrome P-450 and the associated NADH- and NADPH-cytochrome c reductases were assayed, as well as several phase I and phase II enzyme activities. Phase I activities were markers of the families 1, 2, 3 and 4 of P-450; phase II activities were microsomal UDP glucuronosyl transferases (UGT) and cytosolic glutathione S-transferase (GST). Canthaxanthin accumulated in liver to a much higher level than did ingested or injected beta-carotene. Canthaxanthin increased the liver content of cytochrome P-450 (control value x 1.7), and the activity of NADH-cytochrome c reductase (x 1.5), and of some P-450-dependent enzymes (ethoxy-, methoxy-, pentoxy- and benzoxyresorufin O-dealkylases; x98, x15, x6.5 and x13, respectively), but not of others (erythromycin N-demethylase, nitrosodimethylamine N-demethylase and laurate omega-hydroxylase). Phase II activities were also increased: UGT1 (x3.4), UGT2 (x1.2) and GST (x1.2). This induction profile, characterized by the very strong increase of the activity associated with P4501A1, and the co-induction of UGT1, closely resemble that of a classical inducer, 3-methylcholanthrene. By contrast, neither beta-carotene (fed or injected), nor an excess of vitamin A induced any significant variation of the enzyme activities measured.

Effects of a water-soluble extract of rosemary and its purified component rosmarinic acid on xenobiotic-metabolizing enzymes in rat liver.

The effects of a water-soluble extract (WSE) of rosemary and its purified antioxidant rosmarinic acid (RA) on xenobiotic metabolizing enzymes (XME) were studied in rat liver after dietary administration. The modulation of phase I enzymes such as cytochrome P450 (CYP) 1A, 2B, 2E1, 3A, and phase II enzymes such as glutathione S-transferase (GST), quinone reductase (QR) and UDP-glucuronosyltransferase (UGT) was evaluated by measuring enzyme activities with specific substrates. Protein levels of CYPs and rGST A1/A2, A3/A5, M1, M2 and P1 were measured using antibodies in Western blots. Caffeic acid was also studied because it results from RA biotransformation in rat after oral administration. Male SPF Wistar rats received the different compounds at 0.5% (w/w) incorporated into their diet for 2 weeks. WSE, containing RA, flavones and monoterpenes enhanced CYP 1A1, 2B1/2, 2E1 and GST (especially rGST A3/A5, M1 and M2), QR and UGT. On the contrary, no modification of XME was observed in response to RA or CA (except for a slight increase of UGT activity after CA treatment). The induction of XME by WSE could be attributed to flavones, monoterpenes or an additive effect of all components.

Effect of onion consumption by rats on hepatic drug-metabolizing enzymes.

Fruits and vegetables or their natural constituents which increase detoxication enzymes and/or reduce activating enzymes are considered as good candidates to prevent chemically-induced carcinogenesis. In this study, rats were fed a diet supplemented with 20% onion powder for 9 days. Several cytochrome P450 (CYP)s enzymes (CYP 1A, 2B, 2E1, 3A), which are involved in carcinogen activation, were determined by measuring their enzyme activities using specific substrates. In addition, phase II enzymes activities such as UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST), involved in detoxication of carcinogens, were measured. Protein levels of CYPs and GST A1/A2, A3/A5, Ml, M2 and P1 were measured using antibodies in Western blots. Consumption of onion induced CYP 1A and CYP 2B activities while it decreased CYP 2E1 activity. This later modification was accompanied by a decrease of CYP 2E1 levels. The same dietary treatment caused a slight increase of the total GST activity. The relative proportions of GST subunits were modified. GST Al/A2 subunits were increased while GST A3/A5 and GST M2 subunits were decreased and GST M1 and P1 were not modified. Onion consumption also increased p-nitrophenol UGT activity. Taken together, these results suggest that the decrease of CYP 2E1 and the increase of phase II enzymes by onion can afford protection against some carcinogens, while the decrease of some GST subunits could increase the genotoxic effects of other chemicals. The modulating effect of onion could be ascribed to alk(en)yl polysulphides and/or glycosides of flavonols, which were identified in the onion powder.

Effect of prototypic polychlorinated biphenyls on hepatic and renal vitamin contents and on drug-metabolizing enzymes in rats fed diets containing low or high levels of retinyl palmitate.

Two groups of weanling male Sprague-Dawley rats fed a diet supplemented with either 0.6 or 6 retinol equivalents/g diet were each separated into three further groups receiving 300 mumol 2,2',4,4',5,5'-hexachlorobiphenyl/kg body weight, 300 mumol 3,3',4,4'-tetrachlorobiphenyl kg/body weight or vehicle only (corn oil). Only the coplanar (3,4)2Cl congener caused a slight reduction in food intake, thymic atrophy and led to a significant decrease in the liver vitamin A storage. The vitamin A lost by the liver was approximately the same in both dietary groups; however an increased renal accumulation of vitamin A was observed in the high vitamin A group. Serum retinol was reduced by (3,4)2Cl treatment but remained unchanged by (2,4,5)2Cl exposure. Total amounts of ascorbic acid and its oxidation products were increased in the liver and in the kidney by both xenobiotics while niacin and thiamine concentrations were lowered by (3,4)2Cl only. Microsomes from vitamin A-deficient rats exhibited a marked decrease in the anisotropy parameter. After (2,4,5)2Cl exposure, an increase in membrane fluidity was observed linked to a decrease in cholesterol/phospholipid (C/P) ratio. Treatment with (3,4)2Cl caused a significant decrease in the index of fluorescence polarization only in the low vitamin A group even if the C/P ratio was enhanced in both dietary groups. This study shows that the polychlorinated biphenyl with the 3-methylcholanthrene-type pattern of induction of cytochrome P-450 has more profound effects on B group vitamins and particularly vitamin A homeostasis than does the phenobarbital-type inducer. Moreover, this situation, which has been found to be similar to that in vitamin A deficiency, is not ameliorated by a high dietary vitamin A intake.

Post-initiation modulating effects of allyl sulfides in rat hepatocarcinogenesis.

Effects of administration of diallyl sulfide (DAS) and diallyl disulfide (DADS) on the promotion stage of hepatocarcinogenesis were investigated in rats using the Ito model. They were compared with those of phenobarbital (PB), a well-known liver promoter in rats. Initiation was induced by a single dose of N-nitrosodiethylamine (NDEA) and 3 weeks later, a partial hepatectomy was conducted. Two weeks after the NDEA injection, rats received either 0.05% allyl sulfides, PB or both in their diet for 8 weeks. Feeding with DAS increased the number of liver preneoplastic foci by 63% with respect to the untreated group. However, rats fed DAS showed a lower foci development than rats fed PB. The DADS group did not differ from control group for any of the measured morphometric parameters. Simultaneous administration of DADS with PB partially reduced the promotional activity of PB whereas DAS co-treatment did not modify PB properties. These findings confirm that DAS can act as a promoter in rat liver but exerts no co-promoting effect. Conversely, DADS was found to have promotion-inhibiting ability, suggesting that DADS has greater value than DAS as a chemopreventive agent.

Induction of cytochrome P450 and/or detoxication enzymes by various extracts of rosemary: description of specific patterns.

The ability of rosemary to modulate cytochrome P450 (CYP) and detoxication enzymes in rat liver was evaluated by comparing the effects of dried leaves and leaf extracts with different chemical compositions: essential oil (EO) containing monoterpenes, a dichloromethane extract (DCME) containing phenolic diterpenes and a water-soluble extract (WSE) containing phenolic compounds such as rosmarinic acid and flavonoids. Chemical analyses were done in order to characterize the composition of extracts. Male Wistar rats received the leaves or extracts of rosemary in their diet at 0.5% (w/w) for 2 weeks. The effects of such treatments were evaluated for CYP (1A, 2B, 2E1), glutathione S-transferase (GST), NAD(P)H: quinone reductase (QR) and UDP-glucuronosyltransferase (UGT) activities and on protein levels (immunoblot analyses). Expression of specific UGT isoforms (mRNA semi-quantification by RT-PCR) was measured. Our study reports that EO selectively induced CYP, particularly CYP2B. WSE enhanced both CYP and detoxication enzymes. DCME acted as a monofunctional inducer, inducing GST, QR and UGT, in particular UGT1A6. Considering the specific pattern of induction obtained with DCME and WSE treatment, it should be relevant to evaluate the chemopreventive potency of these extracts on carcinogenesis in animal models.

Effects of conjugated linoleic acid isomers on lipid-metabolizing enzymes in male rats.

Male weanling Wistar rats (n = 15), weighing 200-220 g, were allocated for 6 wk to diets containing 1% (by weight) of conjugated linoleic acid (CLA), either as the 9c,11 t-isomer, the 10t,12c-isomer, or as a mixture containing 45% of each of these isomers. The five rats of the control group received 1% of oleic acid instead. Selected enzyme activities were determined in different tissues after cellular subfractionation. None of the CLA-diet induced a hepatic peroxisome-proliferation response, as evidenced by a lack of change in the activity of some characteristic enzymes [i.e., acyl-CoA oxidase, CYP4A1, but also carnitine palmitoyltransferase-I (CPT-I)] or enzyme affected by peroxisome-proliferators (glutathione S-transferase). In addition to the liver, the activity of the rate-limiting beta-oxidation enzyme in mitochondria, CPT-I, did not change either in skeletal muscle or in heart. Conversely, its activity increased more than 30% in the control value in epididymal adipose tissue of the animals fed the CLA-diets containing the 10t,12c-isomer. Conversely, the activity of phosphatidate phosphohydrolase, a rate-limiting enzyme in glycerolipid neosynthesis, remained unchanged in adipose tissue. Kinetic studies conducted on hepatic CPT-I and peroxisomal acyl-CoA oxidase with CoA derivatives predicted a different channeling of CLA isomers through the mitochondrial or the peroxisomal oxidation pathways. In conclusion, the 10t,12c-CLA isomer seems to be more efficiently utilized by the cells than its 9c,11t homolog, though the Wistar rat species appeared to be poorly responsive to CLA diets for the effects measured.