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.     

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.     

Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes

This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC₅₀ = 24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC₅₀ values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.     

Induction of cytochrome P450-dependent monooxygenase in mouse liver and kidney by rutaecarpine, an alkaloid of the herbal drug Evodia rutaecarpa.

Rutaecarpine is one of the main alkaloids of an herbal remedy, Evodia rutaecarpa, which has been used for the treatment of gastrointestinal disorder and headache. Effects of rutaecarpine on hepatic and renal cytochrome P450 (CYP)-dependent monooxygenase were studied in C57BL/6J mice. Treatment of mice with rutaecarpine by gastrogavage at 50 mg/kg/day for three days resulted in 57%, 41%, 6-, and 6-fold increases of hepatic microsomal benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, 7-ethoxyresorufin O-deethylation, and 7-methoxyresorufin O-demethylation activities, respectively. However, the treatment had no effects on hepatic oxidation activities toward benzphetamine, N-nitrosodimethylamine, nifedipine, and erythromycin. In the kidney, rutaecarpine-treatment resulted in 2-fold and 42% increases of microsomal benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation activities, respectively. The treatment also increased renal 7-ethoxyresorufin O-deethylation activity to a detectable level. Immunoblot analysis of microsomal proteins showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine. These CYPs were not detectable in the immunoblot analyses of control and rutaecarpine-treated mouse kidney microsomes. These results indicated that rutaecarpine was a CYP1A inducer and showed potent inductive effects on both CYP1A1 and CYP1A2 in the liver.     

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Induction of drug metabolizing enzymes by 1,7‐phenanthroline and oltipraz in mice is unrelated to Ah‐responsiveness

The protective effect of several classes of compounds against the toxic and neoplastic effects of xenobiotics has been attributed to the induction of noncytochrome P450 (P450) drug metabolizing enzymes. Glutathione S‐transferases (GST), NAD(P)H:quinone oxidoreductase (QOR), and UDP‐glucuronosyltransferases (UGT) play a prominent role in detoxification and can be induced by oltipraz and other N‐heterocyclic compounds in rats. In contrast to the induction of these enzymes by aryl hydrocarbon (Ah)‐receptor agonists, induction by oltipraz and 1,7‐phenanthroline is not accompanied by CYP1A induction. This study investigated the induction of drug metabolizing enzymes following administration of oltipraz and 1,7‐phenanthroline in four mouse strains (C57B6A‐J, Frings × C57B6J, Frings, CF‐1) exhibiting varying degrees of responsiveness to an Ah‐receptor agonist. The relative Ah responsiveness was determined in all strains by the induction of hepatic Cyp1a after three daily doses of 3‐methylcholanthrene (20 mg/kg). After treatment with 1,7‐phenanthroline and oltipraz (150 mg/kg i.g.) daily for 3 days, all strains showed similar induction of GST and QOR activities for each inducer. Both compounds were equally effective in elevating GST activity, but 1,7‐phenanthroline was more effective than oltipraz in elevating QOR activity. In addition to GST and QOR changes, 1,7‐phenanthroline significantly elevated UGT (1‐naphthol) activity in the Frings strain. Neither compound produced significant changes in Cyp1a parameters. The independence of 1,7‐phenanthroline and oltipraz induction of GST and QOR from Cyp1a‐responsiveness is in line with the concept that N‐heterocycle‐containing inducers act by mechanisms other than an Ah‐receptor‐dependent pathway in which the P450 response has been masked or prevented. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 77‐82, 1999     

Modular bioreactor for primary human hepatocyte culture: medium flow stimulates expression and activity of detoxification genes

Down-regulation of detoxification genes, notably cytochrome P450 (CYPs), in primary hepatocyte cultures is a long-standing and major concern. We evaluated the influence of medium flow in this model. Hepatocytes isolated from 12 different liver donors were cultured either in a multichamber modular bioreactor (MCmB, flow rate 250-500 μL/min) or under standard/static conditions, and the expression of 32 genes, enzyme activities and biological parameters were measured 7-21 days later. mRNA expression of genes involved in xenobiotic/drug metabolism and transport, including CYP1A1, 1A2, 2B6, 2C9, 3A4 (and activities for some of them), UDP-glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B7, glutathione S-transferase (GSTα), and multidrug resistance protein 1 (MDR1) and MRP2, were specifically up-regulated by medium flow as compared with static controls in all cultures tested. In 2-week-old cultures, expression of detoxification genes reached levels close to or higher than those measured in freshly isolated hepatocytes. In contrast, CYP2D6 and most of other tested genes were not affected by medium flow. We conclude that medium flow specifically interferes with, and up-regulates, the activity of xenosensors and/or the expression of detoxification genes in primary human hepatocytes. Down-regulation of detoxification genes in conventional (static) cultures is therefore partly a consequence of the absence of medium circulation.     

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.     

Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.     

Relationship between induction of aryl hydrocarbon hydroxylase and de novo synthesis of cytochrome P-448 (P1-450) in mice

Phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzpyrene, 3-methylcholanthrene (3-MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were administered i.p. for 1 or 3 days to genetically “responsive” (C57BL/6J) and genetically “non-responsive” (DBA/2J) mice. 3-MC or benzpyrene stimulated aryl hydrocarbon hydroxylase (AHH) activity in C57BL/6J (B6) mice but not in DBA/2J (D2) mice. TCDD induced AHH activity in both B6 and D2 mice. Time-course studies showed that in the first 12 h after a single injection of 3-MC to B6 mice there was no shift in the reduced cytochrome P-450-CO complex absorption spectra from 450 to 448 nm, although AHH activity increased 4–5 times over (above) that of the control group. The relationship between induction of AHH activity by polycyclic hydrocarbons in B6 mice and the concomitant synthesis of cytochrome P-448 is discussed.     

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.     

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.     

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.     

Malnutrition sequela on the drug metabolizing enzymes in male Holtzman rats

The effect of food restriction on the specific activities of the drug metabolizing enzymes (DME) system was studied in Holtzman male rats by comparing DME activities in 90-day-old control rats fed ad libitum (CO), rats fed 40% restricted food (RF) from the gestation period to the day of sacrifice, and recovered rats (rRF) fed 40% restricted food from period of gestation to 45 days of age and then fed ad libitum until the day of sacrifice. In liver, total cytochrome P450 (CYP) of the RF and rRF groups was higher by approximately 50% and 28%, respectively, than in CO rats. Specific activities of individual CYP monooxygenases (MO) such as CYP2B [7-methoxycoumarin demethylase (MOCD)], CYP1A [aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin deethylase (EORD)], and CYP2E [nitrosodimethylamine demethylase (NDMAd)] were 31, 61, 43, and 56% in RF and 16, 36, 26, and 32% in rRF groups, respectively, more than the CO values. Conjugases such as UDP- glucuronosyltransferases with substrates 3-OH benzo(a)pyrene (UGT1) and 4-hydroxybiphenyl (UGT2) and glutathione S-transferase (GST) with substrate 1-chloro-2,4-dinitrobenzene were higher by 72, 69, and 33% in RF and 28, 38, and 24% in rRF groups, respectively. MO activities (MOCD and EORD) were significantly higher in lung, kidney, and intestine: MOCD by 82, 48, and 45% in RF and 40, 25, and 22% in rRF, respectively; and EORD by 84, 77, and 67% in RF and 40, 33, and 28% in rRF, respectively. However, activity of conjugases (UGT1 and GST) were significantly lower (approximately 35-45%) in RF and rRF rats (approximately 20-30%) than in the CO group in above mentioned extrahepatic tissues. These studies indicate that undernourishment during the period of gestation, weanling, and growth and development of microsomal enzymes produces a sequela of events on the DME in hepatic and extrahepatic tissues that cannot return to the control values even when fed ad libitum.     

Absolute requirement for iron in the development of chemically induced uroporphyria in mice treated with 3-methylcholanthrene and 5-aminolevulinate

Accumulating evidence, including experiments using cytochrome P450 1a2 (Cyp1a2) gene knock-out mice (Cyp1a2(−/−)), indicates that the development of chemically induced porphyria requires the expression of CYP1A2. It has also been demonstrated that iron enhances and expedites the development of experimental uroporphyria, but that iron alone without CYP1A2 expression, as in Cyp1a2(−/−) mice, does not cause uroporphyria. The role of iron in the development of porphyria has not been elucidated. We examined the in vivo effect of iron deficiency on hepatic URO accumulation in experimental porphyria. Mice were fed diets containing low (iron-deficient diet (IDD), 8.5 mg iron/kg) or normal (normal diet (ND), 213.7 mg iron/kg) levels of iron. They were treated with 3-methylcholanthrene (MC), an archetypal inducer of CYP1A, and 5-aminolevulinate (ALA), precursors of porphyrin and heme. We found that uroporphyrin (URO) levels and uroporphyrinogen oxidation (UROX) activity were markedly increased in ND mice treated with MC and ALA, while the levels were not raised in IDD mice with the same treatments. CYP1A2 levels and methoxyresorufin O-demethylase (MROD) activities, the CYP1A2-mediated reaction, were markedly induced in the livers of both ND and IDD mice treated with MC and ALA. UROX activity, supposedly a CYP1A2-dependent activity, was not enhanced in iron-deficient mice in spite of the fact of induction of CYP1A2. We showed that a sufficient level of iron is essential for the development of porphyria and UROX activity.     

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.     

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.     

Cytochrome P-450 mRNA expression in human liver and its relationship with enzyme activity

CYP activity and protein contents have been measured in human liver using different techniques. In contrast, CYP mRNA data are scarce and the relationships between CYP mRNA contents and activities have not been established. These studies deserve further attention because mRNA determinations by RT-PCR require a very small amount of material (e.g., liver needle biopsy) and could provide important data regarding CYP expression regulation. In this study we measured in 12 human liver samples the mRNA contents of 10 CYPs by quantitative RT-PCR and the metabolic activities using specific substrates. mRNA contents and activities showed high correlation coefficients for CYP1A1, CYP1A2, CYP3A4, CYP2D6, and CYP2B6 (0.96, 0.94, 0.69, 0.61, and 0.52, respectively), but no significant correlations were found for CYP2C9, CYP2A6, and CYP2E1. The results suggest that the regulation of CYP1A1, CYP1A2, CYP3A4, CYP2D6, and CYP2B6 expression is essentially pretranslational and that their mRNA levels could allow a good estimate of their activity.