Enhancement of phase II enzyme activity by purpurin resulting in the suppression of MeIQx–DNA-adduct formation in mice

We previously demonstrated using a bacterial system that the antigenotoxic activity of the anthraquinone compounds purpurin and alizarin was due to the suppression of microsomal enzyme activity involved in the activation of mutagens. In the present study we determined the effect of purpurin and alizarin on (i) MeIQx–DNA-adduct formation in mouse tissues and (ii) the activity of phases I and II enzymes in liver fractions, the liver being the target tissue of MeIQx. The amount of MeIQx–DNA adduct formed was determined using ³²P-postlabeling methods. Methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) enzyme activities, which reflect CYP 1A activity, were measured as markers for phase I enzymes, and UDP–glucuronyltransferase (UGT) and glutathione S-transferase (GST) activities were determined as markers for phase II enzymes. Mice fed with a diet containing 0.5% purpurin for 3 days prior to MeIQx administration had 70% fewer MeIQx–DNA adducts in the lung and kidney, and fewer DNA adducts (insignificant, statistically) in the liver compared with mice fed a diet lacking purpurin. MROD and EROD activities in the liver of these mice increased six- and eight-fold, respectively, and were higher than those determined for the control mice within 1 day following commencement of purpurin treatment. These elevated activities were maintained during treatment and declined immediately following removal of purpurin from the diet. GST and UGT activities gradually increased 2.5- and 3-fold, respectively, following purpurin treatment, and were maintained at significantly high levels even after purpurin administration ceased. Alizarin did not significantly affect DNA-adduct formation and enzyme activity, except in the case of UGT. Taken together, our results show that purpurin reduced MeIQx–DNA-adduct formation by maintaining elevated phase II enzyme activities, thereby facilitating accelerated excretion of MeIQx.     

Modulation of drug-metabolizing enzymes by extracts of a herbal medicine Evodia rutaecarpa in C57BL/6J mice

Evodia rutaecarpa is a traditional Chinese medicine used for the treatment of gastrointestinal disorders and headache. To assess the possible drug interactions, effects of methanol and aqueous extracts of E. rutaecarpa on drug-metabolizing enzymes, cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. Treatment of mice with methanol extract by gastrogavage caused a dose-dependent increase of liver microsomal 7-ethoxyresorufin O-deethylation (EROD) activity. In liver, methanol extract at 2 g/kg caused 47%, 7-, 8-, 4-fold, 81% and 26% increases of benzo(a)pyrene hydroxylation (AHH), EROD, 7-methoxyresorufin O-demethylation (MROD), 7-ethoxycoumarin O-deethylation (ECOD), benzphetamine N-demethylation, and N-nitrosodimethylamine N-demethylation activities, respectively. Aqueous extract at 2 g/kg caused 68%, 2-fold, and 83% increases of EROD, MROD, and ECOD activities, respectively. For conjugation activities, methanol extract elevated UGT and GST activities. Aqueous extract elevated UGT activity without affecting GST activity. Immunoblot analyses showed that methanol extract increased the levels of CYP1A1, CYP1A2, CYP2B-, and GSTYb-immunoreactive proteins. Aqueous extract increased CYP1A2 protein level. In kidney, both extracts had no effects on AHH, ECOD, UGT, and GST activities. Three major bioactive alkaloids rutaecarpine, evodiamine, and dehydroevodiamine were present in both extracts. These alkaloids at 25 mg/kg increased hepatic EROD activity. These results demonstrated that E. rutaecarpa methanol and aqueous extracts could affect drug-metabolizing enzyme activities. Rutaecarpine, evodiamine, and dehydroevodiamine contributed at least in part to the increase of hepatic EROD activity by extracts of E. rutaecarpa. Thus, caution should be paid to the possible drug interactions of E. rutaecarpa and CYP substrates.     

Phase I and II liver enzyme activities in juvenile alligators (Alligator mississippiensis) collected from three sites in the Kissimmee-Everglades drainage, Florida (USA)

We examined CYP1A (measured using hepatic EROD and MROD activities) and glutathione-S-transferase (GST) activities in juvenile alligators (Alligator mississippiensis) collected from three sites with varying contamination in the Kissimmee-Everglades drainage in south Florida. We hypothesized that contaminants present in areas with intermediate or higher contaminant concentrations would alter hepatic enzyme activities in juvenile alligators from those sites when compared to hepatic enzyme activity in animals from the area with the least contamination. EROD activity was found to be higher in animals from the site with lower reported levels of contamination relative to those from the site with the highest reported contamination suggesting an inhibition of CYP1A expression or activity. No differences among animals from the three sites were observed for hepatic MROD and GST activities. A significant negative relationship between EROD, MROD, and GST activities and body size was exhibited in alligators from the site with the lowest contamination. No relationship between body size and hepatic enzyme activity was found in animals from the sites with intermediate and higher contamination, suggesting that contaminants present at these sites act to alter this relationship. No correlation was observed in this study between plasma steroid concentrations (estradiol-17 beta or testosterone) and hepatic EROD, MROD, or GST activities.     

Effects of baicalein and wogonin on drug-metabolizing enzymes in C57BL/6J mice

Effects of baicalein and wogonin, the major flavonoids of Scutellariae radix, on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. One-week treatment of mice with a liquid diet containing 5 mM baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic benzo(a)pyrene hydroxylation (AHH), benzphetamine N-demethylation (BDM), N-nitrosodimethylamine N-demethylation (NDM), nifedipine oxidation (NFO), and erythromycin N-demethylation (EMDM) activities, respectively. Treatment with a liquid diet containing 5 mM wogonin resulted in 43%, 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, hepatic 7-methoxyresorufin O-demethylation (MROD) activity was increased and decreased by baicalein- and wogonin-treatments, respectively. Similar modulation was observed with caffeine 3-demethylation (CDM) activity. Immunoblot analysis showed that the levels of hepatic CYP2E1 and CYP3A proteins were decreased by both baicalein- and wogonin-treatments. Hepatic CYP1A2 protein level was increased by baicalein but decreased by wogonin. In extrahepatic tissues, renal AHH activity was decreased by wogonin whereas pulmonary AHH, 7-ethoxyresorufin O-deethylation (EROD), and MROD activities were increased by both flavonoids. Both baicalein and wogonin strongly increased CYP1A protein level in mouse lung. Hepatic and renal UGT activities toward p-nitrophenol were suppressed by baicalein- and wogonin-treatments. However, cytosolic GST activity was not affected by flavonoids. These results suggest that ingestion of baicalein or wogonin can modulate drug-metabolizing enzymes and the modulation shows tissue specificity.     

Longterm stability of phase I and phase II enzymes of porcine liver cells in flat membrane bioreactors

Recently, researchers have focused on the use of bioartificial liver devices to support patients with fulminant hepatic failure. Our team developed a cell-based flat membrane bioreactor (FMB). In this, porcine liver cells were maintained in 3D-coculture between two gel layers in a sandwich configuration for 3 weeks to study the influence of this bioreactor technique on the preservation of basic, not induced activities of phase I and phase II enzymes. First, the time and substrate dependencies of the following enzymes were measured: ethoxyresorufin-O-deethylase (EROD, CYP 1A1/1A2) and ethoxycoumarin-O-deethylase (ECOD, CYP 2B6) as phase I enzymes, and glutathione-S-transferase (GST), UDP-glucuronosyltransferase (UGT) and sulfotransferase (ST) as phase II enzymes. To find optimal test conditions Michaelis-Menten kinetics were calculated. Next, different potential inducers were tested to find out the most effective compounds. Based on these results, the basic, not induced levels of the different enzymes were determined in the flat membrane bioreactor. Furthermore, the response of these enzyme activities to the chosen inducers was investigated to examine whether the cells keep their ability for drug-drug interactions. Basic, not induced activities of both phase I enzymes and the phase II enzymes GST and UGT were maintained at nearly the initial levels during the complete period of study. In addition, it was possible to induce these enzymes twice or three times in a weekly interval. In contrast, the basic, not induced activity of ST increased during the first 10 days of culture. It stabilized then and was maintained steady. As in short-term investigations, no reaction of the ST-activity towards any inducer could be obtained. These results prove that porcine liver cells preserve their phase I and phase II activities and respond to inducing drugs over 3 weeks in culture. Therefore, the flat membrane bioreactor is not only suitable for investigating drug metabolism, drug-drug interactions, and enzyme induction but also for supporting liver functions.     

Activity patterns of biotransformation enzymes in juvenile chickens after in ovo dosage of PCB126

Little is known about the correlations between biotransformation enzymes in juvenile birds after exposure to environmental toxicants like PCBs. In this study eggs of domestic chicken (Gallus domesticus) were dosed with PCB126 in concentrations of 0.175-0.325 ng/g egg weight. Liver subcellular fractions were analyzed for activities of Phase 1 and Phase 2 biotransformation enzymes 2 and 5 weeks post-hatch. Ethoxyresorufin O-deethylase (EROD) activity was increased in both the 2-week and 5-week samples. Glutathione-S-transferase (GST) activity was increased in the 2-week samples only, but the 5-week samples showed an overall much higher GST activity, probably as a result of a still developing enzyme expression in maturing chickens. The same pattern was seen in the phenol-type UDP-glucuronosyltransferase (UGT) activity of the control animals. The week two samples showed a positive dose-response relationship for the UGT activity, but after 5 weeks this was reversed, possibly caused by inhibition of hydroxylated PCB metabolites. Phenol-type sulfotransferase (SULT) activities were not significantly correlated with time or dose. There was a strong positive regression between the Ah-receptor mediated EROD and UGT activities. The EROD activities were also positively correlated to the GST activities. Most interesting was a negative correlation between the UGT and SULT activities: an inhibited UGT activity appeared to be compensated by an increased SULT activity.     

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

Effects of Echinococcus multilocularis metacestodes infection and drug treatment on the activities of biotransformation enzymes in mouse liver

The changes of biotransformation enzymes will substantially affect the host's ability to metabolize drugs and other xenobiotic compounds. In order to further elucidate this process and promote the development in treatment of echinococcosis, we investigated the effects of Echinococcus multilocularis infection and drug treatment on biotransformation enzymes in mouse liver. In microsomal and cytosolic fractions, from the six activities assayed, significant decrease of glutathione S-transferases (GST) activity and significant increase of 7-pentoxyresorufin (PROD) and NADPH-cytochrome P450 reductase (CPR) activity were observed in the mice infected with E. multilocularis metacestodes. In addition, after six weeks treatment of albendazole in E. multilocularis infected mice, significant decreased GST activity and significant increase of 7- ethoxyresorufin (EROD), PROD, and particularly 3-fold higher 7-methoxyresorufin (MROD) activity were observed. The 3-bromopyruvate treated mice only exhibited significantly lower GST activity. Our results demonstrate that E. multilocularis metacestodes infection can affect the activities of main hepatic biotransformation enzymes and such alterations of activity may further affect the hepatic biotransformation of xenobiotics. Moreover, albendazole and 3-bromopyruvate, the promising potential drug against Echinococcus, affected different hepatic biotransformation enzymes and may affect their metabolism. The findings will help to develop rational treatments with less side effects and promote the development of more efficient treatments against E. multilocularis.     

Effect of Siamese cassia leaves on the activities of chemical carcinogen metabolizing enzymes and on mammary gland carcinogenesis in the rat.

Male Wistar rats were fed AIN-76 semipurified diet or diet containing 5% ground lyophilized Siamese cassia leaves for 2 weeks before sacrifice. Hepatic S9 fractions were prepared and assayed for the level of cytochrome P450 (P450), the activities of monooxygenase, i.e., aniline hydroxylase (ANH), aminopyrine-N-demethylase (AMD) as well as the capacity to metabolically activate the mutagenicities of aflatoxin B(1) (AFB(1)) and benzo(a)pyrene (B(a)P). In addition, the activities of detoxificating enzymes such as glutathione-S-transferase (GST) and UDP-glucuronyltransferase (UGT) were also measured. It was found that feeding of Siamese cassia leaves significantly reduced the activities of hepatic ANH and AMD as well as the capacity to activate the mutagenicity of AFB(1) towards Salmonella typhimurium TA100, being 31, 73 and 41% of control group, respectively. It also slightly decreased, but not significantly, the capacity to activate the mutagenicity of B(a)P towards S. typhimurium YG1029. On the other hand, however, the activities of both GST and UGT were markedly increased in those animals, being 250 and 220% of control animals. The anticarcinogenic potential of Siamese cassia leaves was also investigated in female Sprague Dawley rats treated with 9,10-dimethyl-1,2-benzanthracene (DMBA). The animals were fed control diet or diet containing ground lyophilized Siamese cassia leaves 2 weeks prior to and 1 week after intragastrically administration of DMBA, and then they were placed on a pellet diet for additional 25 weeks. Interestingly, it was found that feeding of diet containing 2.5 and 4% Siamese cassia leaves resulted in a significant decrease in the multiplicity of mammary gland tumors as well as a slight delay of the onset of tumor development. The incidence of tumors in the group fed 4% Siamese cassia leaves, but not in the 2.5% group, was lowered, although not significantly, than that of control group. The results in the present study therefore demonstrated that Siamese cassia leaves possess phase II enzyme inducing property as well as the ability to reduce some phase I enzyme activities in rat liver. This Thai vegetable also exhibit cancer chemopreventive potential, at least against DMBA-induced mammary gland carcinogenesis which may be partly due to phase II inducing capacity as well as phase I inhibitory activity.     

Antioxidant enzymes in the liver of Chelidonichthys obscurus from the Montenegrin coastline

The activities of antioxidant defence enzymes — total, manganese and copper zinc containing superoxide dismutase (Tot SOD, Mn SOD, CuZn SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and biotransformation phase II enzyme glutathione-S-transferase (GST) — in the liver of longfin gurnard (Chelidonichthys obscurus) from the Montenegrin coastline (Adriatic sea) were investigated. The specimens were collected in winter (February) and late spring (May) at two localities: Platamuni (PL, potentially unpolluted) and the Estuary of the River Bojana (EB, potentially polluted). The obtained results show that the activities of Mn SOD, CAT, GSH-Px and GST in winter were significantly lower at EB than at PL. In spring, the activities of CAT and GST were decreased, while GR activity was increased at EB in comparison to PL. The activities of Mn SOD and GST at PL were decreased and GSH-Px, GR and GST activities at EB were increased in spring compared to winter. Our work represents the first study of liver antioxidant enzymes of longfin gurnard from the Montenegrin coastline and reveals that locality, as a variable, has a greater influence on antioxidant enzymes and biotransformation phase II enzyme GST activities compared to season.     

Induction of cytochrome P450 1A1 and conjugating enzymes in rainbow trout (Oncorhynchus mykiss) liver: A time course study

1. The effects of i.p. injections of isosafrole (ISF) or β-naphthoflavone (β-NF) on the cytochrome P450 (CYP) 1A1 system and conjugating enzymes were investigated in livers from juvenile rainbow trout in a time course study employing catalytic, immunochemical and cDNA probes.2. β-NF treatment resulted in a rapid rise in CYP1A1 mRNA followed by accumulation of P450 1A1 protein and P450 1A1 mediated enzyme activity measured as ethoxyresorufin-O-deethylase (EROD) activity.3. ISF treatment resulted in a comparatively weak induction of CYP1A1 mRNA and P450 1A1 protein levels whilst EROD activity was markedly induced; thus when expressed on the basis of immunoquantified P450 1A1 protein, the specific EROD activity was signficantly higher in ISF than β-NF treated fish.4. In vitro inhibition studies revealed that ISF inhibited EROD activity to a far lesser extent than β-NF.5. Conjugation enzymes represented by phenol UDP-glucuronosyltransferase and glutathione S-transferase (GST) activities, were induced by β-NF, whereas ISF treatment had no effect on these enzyme activities.6. Immunoblotting using antibodies raised against rat GST7-7 showed that a Pi class trout GST enzyme was induced by β-NF treatment.     

Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method

DNA adducts formed by 12 heterocyclic amines were analyzed by 32P-postlabeling method. Several DNA adducts were detected in rat liver by administration of each heterocyclic amine. Total adduct levels ranged from 0.5 for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to more than 250 for 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) per 10(7) nucleotides 24 hr after intragastric administration of these compounds. The N-hydroxy derivative of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was reactive toward DNA in vitro to form adducts. Addition of acetic anhydride to N-OH-MeIQx greatly enhanced its reactivity to DNA. 32P-Postlabeling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx would be metabolized in vivo to N-hydroxy form and further esterified to produce more reactive species, such as N-acetoxy form, which modify DNA to form adducts.     

Drug-metabolizing enzyme activities in freshly isolated oval cells and in an established oval cell line from carcinogen-fed rats

The activities of several different phase I and phase II drug-metabolizing enzymes were measured in freshly isolated oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 weeks and alsoin vitro in the established oval cell line OC/CDE 6. No cytochrome P450 was spectrophotometrically measurable in both preparations and two cytochrome P450-dependent monoxygenase activities, aminopyrineN-demethylase and ethoxyresorufinO-deethylase, could not be detected in the oval cells of both sources. However, cytosolic glutathione transferase, microsomal expoxide hydrolase and UDP-glucuronosyltransferase activities were clearly measurable in oval cells. Similar enzyme activities were found in freshly isolated and cultured oval cells. The highest activities of these three enzymes were detected during the exponential growth phase of the cultured cells; thereafter the activities decreased until the cells reached confluency. Changes in phenol UDP-glucuronosyltransferase (UGT1A1) mRNA levels paralleled the variations in UDP-glucuronosyltransferase activity, i.e. they were high in exponentially growing oval cells and low in confluent cell cultures. Taking into account that oval cells are able to proliferate in the livers of rats continuously fed a choline-deficient/DL-ethionine-supplemented diet and that none of the analyzed drug metabolizing enzymes are involved in the activation or detoxication ofDL-ethionine, the described pattern might be part of a more general, nonspecific, protection mechanism enabling these cells to overcome the cytotoxic effects of a variety of carcinogens and to proliferate even in their presence. Furthermore, the expression of microsomal epoxide hydrolase, cytosolic glutathione transferase and UDP-glucuronosyltransferase appears to depend on the proliferative status of the cells.Abbreviations CDE choline-deficient/DL-ethionine-supplemented diet - GST glutathione transferase - mEH microsomal epoxide hydrolase - UGT UDP-glucuronosyltransferase     

Garlic Oil Attenuated Nitrosodiethylamine-Induced Hepatocarcinogenesis by Modulating the Metabolic Activation and Detoxification Enzymes

Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes.     

The effects of diet composition and ration on biotransformation enzymes and stress parameters in rainbow trout, Oncorhynchus mykiss

Juvenile rainbow trout (Oncorhynchus mykiss) were fed one of three isoenergetic diets varying in protein (35-55%) and lipid content (8-18%), at full satiation levels or half rations for 6 weeks in order to investigate the effects of diet on baseline stress parameters and biotransformation enzyme activity. Growth was greatest in fish fed to satiation on a low protein and high lipid diet. Stress parameters, including plasma lactate, glucose and cortisol concentrations were not significantly affected by dietary treatment or ration. Basal biotransformation enzymes, including glutathione S-transferase (GST) and ethoxyresorufin O-deethylase (EROD) activity, were also unaffected by dietary treatment. Fish exposed to the biotransformation enzyme inducer beta-naphthoflavone did not exhibit an alteration in stress indicators or GST activity; however, EROD activity was increased (87- to 210-fold) in fish receiving all diets and rations. The results of the present study indicate that, unlike mammals, fish may be more recalcitrant to different levels of ingestion of isoenergetic diets varying in protein and lipid concentration with respect to stress responses and the maintenance of basal titers of biotransformation enzymes and their induction.     

Differential inhibition of hepatic microsomal alkoxyresorufin O-dealkylation activities by tetrachlorobiphenyls

Polychlorinated biphenyls (PCBs) elicit a spectrum of biochemical and toxic effects in exposed animals. In the present study, we assessed the effect of PCB structure, using four symmetrically-substituted PCBs, on cytochrome P450 (CYP)-mediated methoxy-, ethoxy- and benzyloxyresorufin O-dealkylase (MROD, EROD and BROD, respectively) activities. We found that 2,2',4,4'-tetrachlorobiphenyl (PCB 47), 2,2',5,5'-tetrachlorobiphenyl (PCB 52), 2,2',6,6'-tetrachlorobiphenyl (PCB 54) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) inhibited alkoxyresorufin O-dealkylase activities in hepatic microsomes from 3-methylcholanthrene (MC) or phenobarbital (PB)-treated rats. Measurement of the in vitro inhibitory potencies of the tetrachlorobiphenyls revealed that MROD, EROD and BROD activities were differentially inhibited and the degree of inhibition was determined by the chlorination pattern of the PCB. PCB 77 was more potent than PCB 47 or PCB 52 at inhibiting MROD and EROD activities in hepatic microsomes from MC-treated rats, while no inhibition of either activity was observed with PCB 54. In contrast, BROD activity measured in hepatic microsomes from PB-treated rats was inhibited by PCB 47, PCB 52 and PCB 54 but not by PCB 77. The mode of inhibition for each activity was also evaluated statistically. Inhibition of the alkoxyresorufin O-dealkylase activities could not be discerned in hepatic microsomes from corn oil-treated rats because the activities were inherently too low. No evidence for mechanism-based inhibition of MROD, EROD or BROD activities or an effect via CYP reductase was found. The results demonstrate that relatively coplanar PCBs such as PCB 77 preferentially inhibit EROD and MROD activities, whereas noncoplanar PCBs such as PCB 54 preferentially inhibit BROD activity.     

Anti-genotoxic activity of Vitis coignetiae Pulliat towards heterocyclic amines and isolation and identification of caftaric acid as an antimutagenic component from the juice

Our study demonstrated that the formation of DNA adducts in liver, lungs, colon and kidneys of mice given a carcinogenic heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), in the diet significantly decreased following the administration of the juice of Vitis coignetiae, purple berries from a vine tree. The juice of V. coignetiae significantly inhibited the clastogenicity and mutagenicity of heterocyclic amines in the micronucleus assay and the Ames test, and was an effective inhibitor of the activities of phase I enzymes (cytochrome P450 1A1 and cytochrome P450 1A2) and enhancer of the activities of phase II enzymes (uridine 5'-diphospho-glucuronosyltransferase and glutathione S-transferase). We investigated the purification and isolation of an active compound in the juice of V. coignetiae using antimutagenicity as a separation marker. Caftaric acid, a polyphenolic compound, was identified as a component responsible for antimutagenicity in the juice of V. coignetiae towards the carcinogenic heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). This is the first report of antimutagenicity of caftaric acid. Caftaric acid was reported as an inhibitor of the protein-protein interactions mediated by the Src-family kinases. The impact of the juice of V. coignetiae and its constituents on tumor initiation and promotion thus warrants further study.     

The in vitro metabolism and bioactivation of 1,2-dibromoethane (ethylene dibromide) by human liver

The nematocide, grain fumigant, and gasoline additive 1,2-dibromoethane (DBE) is both a cellular and a genetic toxin that is metabolically activated in rats and mice by mixed function oxidases (MFO) as well as glutathione 5-transferases (GST). The purpose of this study was to determine whether DBE is similarly metabolized and bioactivated by human liver in vitro. Human liver microsomal and cytosolic metabolism of DBE was monitored by the production of aqueous-soluble metabolites from [14-C]-DBE. Reactive intermediates were detected as irreversibly bound adducts to protein or DNA. 1,2-Dibromoethane was metabolized by human liver cytosolic GST, microsomal GST, and microsomal MFO. Cytosolic GST activity (9 +/- 2 nmol/20 min/mg protein) was about four times greater than the other two activities. Only MFO activity resulted in adducts irreversibly bound to protein (1.5 +/- .4 nmol/20 min/mg protein) and was inhibited by the presence of glutathione. Both MFO and GST activity resulted in irreversibly bound adducts to DNA. Microsomal and cytosolic GST activity each produced about twice as many DNA adducts as microsomal MFO activity. These results suggest that human liver, like rat and mouse liver, metabolizes DBE to aqueous-soluble metabolites by both MFO and GST activity. Furthermore, each of these activities produces reactive metabolites that can irreversibly bind to cellular macromolecules.