Decreased hepatic drug metabolising enzyme activity in rats with nitrosamine-induced tumours

N-Methyl N-benzyl nitrosamine (MBNA), which requires P450-dependant activation to be mutagenic, has been shown to produce squamous cell carcinoma of rat oesophagus. The aim of this study was to determine the effects of tumour induction on hepatic cytochrome P450 (CYP) and phase II enzyme activity. Female Wistar rats were given MBNA (2.5 mg/kg) by gavage, twice weekly for 12 weeks. At the end of 12 weeks they were sacrificed; livers and oesophagi were removed. The activity of hepatic CYP and phase II enzymes was determined by incubation of liver microsomes with appropriate CYP substrates. All rats receiving MBNA developed oesophageal lesions. Hepatic CYP1A2 activity (phenacetin 5 microM) in tumour-bearing rats was significantly decreased to 53% of the controls (p <0.05). CYP2E1 (p-nitrophenol hydroxylase), CYP2D (debrisoquine hydroxylase) and CYP3A (quinine hydroxylase) activity was significantly (p <0.05) reduced. Microsomal UDP-glucuronosyl transferase activity was also found to be markedly decreased while glutathione-S-transferase activity remained almost unchanged. Alteration of the activities of drug metabolising enzymes in rats with chemically induced tumours could be an important factor in determining resistance or susceptibility to xenobiotics and antitumour drugs.     

Activities of xenobiotic metabolizing enzymes in rat placenta and liver in vitro

In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling.     

Effect of herbal teas on hepatic drug metabolizing enzymes in rats.

We have investigated the effect of herbal teas (peppermint, chamomile and dandelion) on the activity of hepatic phase I and phase II metabolizing enzymes using rat liver microsomes. Female Wistar rats were divided into six groups (n = 5 each). Three groups had free access to a tea solution (2%) while the control group had water. Two groups received either green tea extract (0.1%) or aqueous caffeine solution (0.0625%). After four weeks of pretreatment, different cytochrome P450 (CYP) isoforms and phase II enzyme activities were determined by incubation of liver microsomes or cytosol with appropriate substrates. Activity of CYP1A2 in the liver microsomes of rats receiving dandelion, peppermint or chamomile tea was significantly decreased (P < 0.05) to 15%, 24% and 39% of the control value, respectively. CYP1A2 activity was significantly increased by pretreatment with caffeine solution. No alterations were observed in the activities of CYP2D and CYP3A in any group of the pretreated rats. Activity of CYP2E in rats receiving dandelion or peppermint tea was significantly lower than in the control group, 48% and 60% of the control, respectively. There was a dramatic increase (244% of control) in the activity of phase II detoxifying enzyme UDP-glucuronosyl transferase in the dandelion tea-pretreated group. There was no change in the activity of glutathione-S-transferase. The results suggested that, like green and black teas, certain herbal teas can cause modulation of phase I and phase II drug metabolizing enzymes.     

Chronic administration of caderofloxacin,a new fluoroquinolone,increases hepatic CYP2E1 expression and activity in rats

Aim:

Caderofloxacin is a new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of caderofloxacin interacting with co-administered drugs.

Methods:

Male rats were treated with caderofloxacin (9 mg/kg, ig) once or twice daily for 14 consecutive days. The effects of caderofloxacin on CYP3A, 2D6, 2C19, 1A2, 2E1 and 2C9 were evaluated using a “cocktail” of 6 probes (midazolam, dextromethorphan, omeprazole, theophylline, chlorzoxazone and diclofenac) injected on d 0 (prior to caderofloxacin exposure) and d 15 (after caderofloxacin exposure). Hepatic microsomes from the caderofloxacin-treated rats were used to assess CYP2E1 activity and chlorzoxazone metabolism. The expression of CYP2E1 mRNA and protein in hepatic microsomes was analyzed with RT-PCR and Western blotting, respectively.

Results:

Fourteen-day administration of caderofloxacin significantly increased the activity of hepatic CYP2E1, leading to enhanced metabolism of chlorzoxazone. In vitro microsomal study confirmed that CYP2E1 was a major metabolic enzyme involved in chlorzoxazone metabolism, and the 14-d administration of caderofloxacin significantly increased the activity of CYP2E1 in hepatic microsomes, resulting in increased formation of 6-hydroxychlorzoxazone. Furthermore, the 14-d administration of caderofloxacin significantly increased the expression of CYP2E1 mRNA and protein in liver microsomes, which was consistent with the pharmacokinetic results.

Conclusion:

Fourteen-day administration of caderofloxacin can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a potential drug-drug interaction mediated by CYP2E1 induction should be considered.     

Changes in hepatic phase I and phase II biotransformation enzyme expression and glutathione levels following atrazine exposure in female rats

1. To determine the effects of repeated atrazine (ATR) treatment on hepatic phase I and II enzymes, adult female rats were treated with vehicle or 100?mg/kg of ATR for 1, 2, 3 or 4 days. Glutathione-s-transferases (GST) mRNA expression, protein levels (mu, pi, alpha, omega), and activity (cytosolic and microsomal), along with bioavailable glutathione (GSH) were assayed.

2. GST expression, concentrations and activity were increased, along with GSH levels, in animals treated with ATR for 3 and 4 days.

3. A subsequent study was performed with animals treated with vehicle, 6.5, 50 or 100?mg/kg/day for 4, 8 or 14 days. Expression of hepatic phase I CYP 450 enzymes was evaluated in conjugation with GST expression, protein and activity. Nineteen of the 45 CYP enzymes assayed displayed increased mRNA levels after eight?days of treatment in animals treated with 50 or 100?mg/kg/day. After 14 days of treatment, all CYP expression levels returned to control levels except for CYP2B2, CYP2B3, CYP2C7, CYP2C23, CYP2E1, CYP3A9, CYP4A3 and CYP27A1, which remained elevated.

4. Results indicate that there may be a habituation or adaptation of liver phase I and phase II expression following repeated ATR treatment.     

Effect of donor age on the levels of activity of rat, hamster and human liver S9 preparations in the Salmonella mutagenicity assay

Liver S9 fractions were prepared from male and female Syrian Golden hamsters and Sprague-Dawley rats, 1, 3, 6 and 12 months of age, which were either uninduced (corn-oil treated) or induced with Aroclor 1254 suspended in corn oil. These preparations were compared at varying protein levels for their ability to metabolize polycyclic aromatic hydrocarbons (benzo(a)pyrene, 3-methylcholanthrene, 7,12-dimethylbenzanthracene), aromatic amines (N-2-acetyl-aminofluorene, beta-naphthylamine, benzidine), and nitroso compounds (N-nitroso-diethylamine, nitrosopyrrolidine, nitrosodiethylmethylurea) to products mutagenic to Salmonella typhimurium. With 3-methylcholanthrene or benzo(a)pyrene in the presence of S9 preparations from Aroclor-treated male rats, the numbers of revertant colonies decreased with increasing age of the animals. Mutagenicity of aromatic amines was not affected by the age of the donor animals from which the S9 was prepared. The use of liver S9 from 1-month-old hamsters produced the highest number of revertant colonies with nitrosodiethylamine. This number decreased with preparations from animals of increasing age. The greatest number of revertant colonies with nitrosopyrrolidine occurred with preparations from male hamsters. A decrease in numbers of revertant colonies with increasing age was observed with the S9 preparation from Arcolor-treated male rats. Nitroso-diethylmethylurea was mutagenic only in the presence of S9 from male or female Aroclor-treated hamsters and the metabolic activity of the S9 preparations did not change with age. S9 preparations from livers of 50-70-year-old humans were compared for their ability to produce mutagenic metabolites at a number of protein levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of tannic acid on cytochrome P450 and phase II enzymes in mouse liver and kidney

Tannic acid, a naturally occurring plant polyphenol, was shown to decrease the mutagenicity and/or carcinogenicity of several amines derivatives and polycyclic aromatic hydrocarbons in rodents. The aim of this study was to evaluate the effect of tannic acid on the activities of murine cytochrome P450 and phase II enzymes. The activities of ethoxyresorufin-O-deethylase (EROD), methoxyresorufin-O-demethylase (MROD), p-nitrophenol hydroxylase (PNPH), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UDPGT) and NAD(P)H:quinone oxidoreductase (NQO1) were measured in the liver and kidney microsomes of female Swiss mice treated intraperitoneally (i.p.) with tannic acid in the dose range of 20-80 mg/kg. At the highest dose, tannic acid decreased the activities of EROD and MROD by 25-28% in mouse liver, while the activity of both hepatic and renal PNPH was reduced by approximately 50% as result of treatment. Moreover, Western blot analysis with CYP2E1 specific antibody showed a significant decrease in the levels of hepatic CYP2E1 in tannic acid treated animals. This polyphenol affected also the phase II enzymes in both tissues examined. The activity of GST was elevated in kidneys, but reduced in livers of the animals treated with tannic acid. The most striking effect was the inhibition of hepatic NQO1. The effect was dose dependent and almost 90% inhibition was observed after the treatment with tannic acid at the dose of 60 and 80 mg/kg. The same treatment caused the approximately 60% inhibition of renal NQO1. These results indicate that tannic acid, beside of scavenging active metabolites of chemical carcinogens, can change their metabolism by modulating the enzymes involved in xenobiotics activation and/or detoxification pathways.     

Fipronil induces CYP isoforms in rats

The goal of the present study was to evaluate fipronil effects on the activities of drug metabolizing enzymes in rat liver microsomes. Rats were orally treated with fipronil at doses of 1, 5, 10 and 15 mg/kg bw/day for 6 days. Determinations of cytochrome P450 (CYP) enzyme activities were carried out in hepatic microsomes isolated from treated rats. The activities of some members of CYP2E, CYP1A, CYP2A, CYP2B and CYP3A subfamilies significantly increased after fipronil treatment in a dose-dependent manner as compared to control. The major effects were observed in the O-deethylation of ethoxyresorufin and O-demethylation of methoxyresorufin (reflecting CYP1A1/2 activities), in the O-depenthylation of pentoxyresorufin and 16β-hydroxylation of testosterone (reflecting CYP2B1/2 activities), and in the N-demethylation of erythromycin and 6β-hydroxylation of testosterone (reflecting CYP3A1/2 activities). Immunoblot studies revealed that fipronil increased the apoprotein levels of CYP1A1. Our results suggest that fipronil is an inducer of hepatic phase I CYP enzymes, causing an increased potential to interact with a wide range of xenobiotics or endogenous chemicals that are substrates of the CYP1A, CYP2B and CYP3A subfamilies. Further investigations are required to in vivo evaluate the potential of the metabolite fipronil sulfone as an inducer of phase I CYP enzymes.     

Altered hepatic drug-metabolizing activity in rats suffering from hypoxemia with experimentally induced acute lung impairment

1.?Hepatic drug-metabolizing activity was investigated in vitro with liver microsomes prepared from rats suffering from hypoxemia with experimentally induced acute lung impairment (ALI).

2.?Male Wistar rats received an intrabronchial administration of dilute hydrochloride solution for ALI induction. Pooled liver microsomes were prepared for the normal and ALI rats, and the hepatic drug metabolism mediated by cytochrome P450 (CYP) 3?A was examined in an incubation study with the microsomes.

3.?The NADPH-dependent metabolism of midazolam significantly increases in ALI rats as compared with that in normal rats. Testosterone 6β-hydroxylation was also observed to significantly increase in ALI rats.

4.?When the hepatic expression of CYP3A proteins was examined, the protein expression of CYP3A1 was shown to significantly increase and that of CYP3A2 remained unaltered in ALI rats. The hepatic expression of NADPH-cytochrome P450 reductase (POR), a protein mediating electron transfer in CYP-mediated drug metabolism, was also revealed to significantly increases in ALI rats.

5.?With the findings regarding the midazolam elimination, the hepatic drug-metabolizing activity seems to increase in response to acute hypoxemia, partly due to an altered expression of the CYP3A enzymes, and an augmented electron transfer with an increased POR expression is probably involved in the increase.     

Induction of hepatic xenobiotic metabolizing enzymes in female Fischer-344 rats following repeated inhalation exposure to decamethylcyclopentasiloxane (D5).

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane with a wide range of commercial applications. The present study was designed to investigate the effects of D5 on the expression and activity of selected rat hepatic phase I and phase II metabolizing enzymes. Female Fischer-344 rats were exposed to 160 ppm D5 vapors (6 h/day, 7 days/week, for 28 days) by whole-body inhalation. Changes in the activity and relative abundance of hepatic microsomal cytochromes P450 (CYP1A, CYP2B, CYP3A, and CYP4A), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were measured. Repeated inhalation exposure of rats to D5 increased liver size by 16% relative to controls by day 28. During a 14-day post-exposure period, liver size in D5-exposed animals showed significant recovery. Exposure to D5 did not change total hepatic P450, but increased the activity of hepatic NADPH-cytochrome c reductase by 1.4-fold. An evaluation of cytochrome P450 (CYP) enzymes in hepatic microsomes prepared from D5-exposed rats revealed a slight (1.8-fold) increase in 7-ethoxyresorufin O-deethylase (EROD) activity, but no change in immunoreactive CYP1A1/2 protein. A moderate increase (4.2-fold) in both 7-pentoxyresorufin O-depentylase (PROD) activity and immunoreactive CYP2B1/2 protein (3.3-fold) was observed. Testosterone 6beta-hydroxylase activity was also increased (2.4-fold) as was CYP3A1/2 immunoreactive protein. Although a small increase in 11- and 12-hydroxylation of lauric acid was detected, no change in immunoreactive CYP4A levels was measured. Liver microsomal epoxide hydrolase activity and immunoreactive protein increased 1.7- and 1.4-fold, respectively, in the D5-exposed group. UDPGT activity toward chloramphenicol was induced 1.8-fold, while no change in UDPGT activity toward 4-nitrophenol was seen. These results suggest that the profile for enzyme induction following inhalation exposure of female Fischer-344 rats to D5 vapors is similar to that reported for phenobarbital, and therefore D5 may be described as a weak "phenobarbital-like" inducer.     

Species difference in the induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, by 2-methoxy-4-nitroaniline among rats, mice, and guinea pigs

Species difference in the induction of hepatic cytochrome P450 CYP1A subfamily enzymes by 2-methoxy-4-nitroaniline (2-MeO-4-NA) was investigated among male F344 rats, C57BL/6 Cr mice, and Hartley guinea pigs. All species of animals were treated with a single ip injection of 2-MeO-4-NA (0.44 mmol/kg body weight), and changes in levels of the mRNA and protein of hepatic cytochrome P4501A (CYP1A) subfamily enzymes were examined by the methods of RT-PCR and Western blot, respectively. In addition, hepatic microsomal enzyme activities were measured using methoxyresorufin and ethoxyresorufin as substrates of CYP1A2 and CYP1A1, respectively. The overall results of the RT-PCR, Western blot, and measurement of the enzyme activity indicated that 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, occurred only in rats but not any other species of animals examined and that the species difference in the CYP1A induction was not necessarily correlated with that in pharmacokinetics of 2-MeO-4-NA. Furthermore, a luciferase reporter gene assay for screening of the ligands of arylhydrocarbon receptor (AhR) using a rat hepatic cell line suggested that 2-MeO-4-NA is not an AhR ligand. The present findings demonstrate for the first time the species difference in the 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes between rats and other rodents, mice and guinea pigs, and further propose an AhR-independent pathway for 2-MeO-4-NA-mediated induction in rats.     

Metabolism of nonylphenol by rat and human microsomes

The in vitro metabolism of [¹⁴C]-nonylphenols (NPs) by rat hepatic microsomes in vitro was examined. Product formation was NADPH dependent and inhibited by the cytochrome P450 inhibitors, piperonyl butoxide and SKF525. Hepatic microsomes isolated from various inducer-treated rats (including β naphthoflavone, phenobarbital, ethanol, dexamethasone, and clofibrate which selectively induce CYP1A, 2B, 2E, 3A and 4A, respectively) all metabolized NPs. Only microsomes from phenobarbital-treated rats exhibited a significantly higher activity towards NPs and showed a different profile of NP metabolites compared to control, untreated rats. Microsomes from human CYP2B6 transfected cells with endogenous NADPH-P450 reductase activity but not microsomes from the non-transfected parent cells metabolized NPs. The metabolism of NPs using microsomes from phenobarbital-treated rats was inhibited by 4-amino-2, 6-dinitro-1-t-butylxylene, a specific CYP2B enzyme inhibitor. Addition of a general anti-CYP2B sera to the reaction mixture attenuated the enzyme activity of microsomes from phenobarbital-treated rats to metabolize NPs. This metabolic reaction was, however, insensitive to a specific anti-CYP2B1 sera that had been shown to inhibit enzyme activities attributed only to CYP2B1 suggesting that the CYP2B2 pathway is predominant in NP metabolism. The results indicate that hepatic cytochrome P450 enzyme(s) can metabolize NPs and that CYP2B isozymes are probably involved.     

Renal and hepatic microsomal enzymes responsible for bioactivation of 3-methoxy-4-aminoazobenzene in the rodent

Activities of the renal and hepatic microsomal enzymes responsible for the N-hydroxylation and mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) were examined in male mice, rats, hamsters and guinea pigs. In all these rodent species, hepatic microsomes showed definite N-hydroxylation of 3-MeO-AAB, whereas the renal activity was detected only in mice. The hepatic enzyme responsible for N-hydroxylation of 3-MeO-AAB (3-MeO-AAB N-hydroxylase) was induced in all species except mice by phenobarbital and selectively in mice and hamsters by 3-methylcholanthrene, whereas these cytochrome P450 inducers did not affect the renal enzyme in mice, rats or hamsters. In individual microsome samples, activities for N-hydroxylation and mutagenic activation of 3-MeO-AAB correlated well. These results indicate that the renal and hepatic enzymes responsible for the metabolic activation of 3-MeO-AAB differed among different species of rodent animals in terms of their activity and inducibility with cytochrome P450 inducers.     

Astaxanthin can alter CYP1A-dependent activities via two different mechanisms: Induction of protein expression and inhibition of NADPH P450 reductase dependent electron transfer

Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase I metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax-containing oil (100 mg Ax/kg body weight/day for 3 days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-, 8.5-, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax-containing oil. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax-containing oil intake can alter CYP1A-dependent activities through two different mechanisms: (1) induction of CYP1A1 mRNA, protein expression, and activity; and (2) inhibition of the electron supply for the enzyme.     

Subacute effects of the brominated flame retardants hexabromocyclododecane and tetrabromobisphenol A on hepatic cytochrome P450 levels in rats

The brominated flame retardants tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are found in the environment, e.g., in sediments and organisms, in food items, human blood samples and mother's milk. In this study, the effects of both compounds on rat hepatic cytochrome P450 (CYP) levels and activities were investigated. Juvenile/young male and female Wistar rats were treated orally with various doses via the feed (TBBPA) or by gavage (HBCD). After 28 days of treatment the animals were sacrificed and hepatic mRNA and microsomes were isolated. HBCD treatment led to a significant induction of CYP2B1 mRNA, CYP2B1/2B2 protein and 7-pentoxyresorufin O-depentylase (PROD) activity suggesting a phenobarbital-type of induction. Furthermore, a significant increase in CYP3A1/3A3 mRNA, CYP3A1 protein, and luciferin benzylether debenzylase (LBD) activity was found, being more pronounced in females than in males. The effect on CYP3A1/3A3 mRNA was significant in female rats at a daily dose of 3.0mg/kg body weight and above. HBCD exhibited no effects on CYP1A2 mRNA, CYP1A1/1A2 protein, or microsomal 7-ethoxyresorufin O-deethylase (EROD) activity suggesting lack of activation of the aryl hydrocarbon receptor. No significant effects on any of the parameters measured were obtained with TBBPA. Our findings suggest that oral exposure to HBCD induces drug-metabolising enzymes in rats probably via the CAR/PXR signalling pathway. Induction of CYPs and co-regulated enzymes of phase II of drug metabolism may affect homeostasis of endogenous substrates including steroid and thyroid hormones.     

Modulation of cytochrome P450 and phase II enzymes by protocatechuic acid in mouse liver and kidney

Protocatechuic acid, a naturally occurring plant polyphenol, was shown to decrease the mutagenicity and/or carcinogenicity of several amine derivatives and polycyclic aromatic hydrocarbons in rodents. In this study the effect of protocatechuic acid on murine cytochrome P450 and phase II enzymes was evaluated. The activities of EROD, MROD, PROD, PNPH, GST, UDPGT and NQO1 were measured in the liver and kidney microsomes of female Swiss mice treated intraperitoneally (i.p.) with protocatechuic acid in the dose range of 80-800 mg/kg. At the highest doses, protocatechuic acid decreased the activities of EROD and MROD by approximately 20-30% in mouse liver and kidney, while the activity of renal PNPH was reduced by 28%. Moreover, Western blot analysis with CYP1A1/1A2 and CYP2E1-specific antibodies showed the same effect on the levels of hepatic CYP1A1/1A2 and CYP2E1 proteins. This simple phenol affected also the phase II enzymes. The activity of GST was elevated in both tissues of the animals treated with protocatechuic acid at the dose of 80 mg/kg. The inhibition of hepatic NQO1 was the most striking effect. The effect was dose dependent and almost 70% inhibition was observed after treatment with protocatechuic acid at the dose of 800 mg/kg. In contrast to the liver, the renal NQO1 was not affected. These results indicate that protocatechuic acid, as other phenolic acids, beside of scavenging active metabolites of chemical carcinogens, can change their metabolism by modulating the enzymes involved in xenobiotics activation and/or detoxification pathways, but this effect depends on tissue.     

Effects of chitosan oligosaccharides on drug-metabolizing enzymes in rat liver and kidneys

To investigate the effect of chitosan oligosaccharides (COS) on drug-metabolizing enzymes in rat liver and kidneys, male Spraque–Dawley rats were fed a diet containing 1% or 3% COS for 5 weeks. The activities of cytochrome P450 (CYP) enzymes, UDP-glucurosyltransferase (UGT) and glutathione S-transferase (GST) in the liver and kidneys were determined. Significant decreases in microsomal CYP3A-catalyzed testosterone 6β-hydroxylation, CYP2C-catalyzed diclofenac 4-hydroxylation, and CYP4A-catalyzed lauric acid 12-hydroxylation in the liver of rats fed the COS diets were observed compared with those rats fed the control diet. Immunoblot analyses of CYP proteins showed the same trend as with enzyme activities. Increased glutathione content in liver was found in rats fed the 1% COS diet. Increased hepatic NADPH: quinone oxidoreductase 1 (NQO1) activity was found in rats fed the COS diets. In kidneys, COS had little or no effect on CYP enzyme activities. However, increased GST activity was observed in rats fed the COS diets. Moreover, a higher UGT activity was found in rats fed the 1% COS diet. Our results indicate that COS may suppress hepatic CYP enzymes and induce phase II detoxifying reactions in the liver and kidneys of rats.     

Multiple cytochrome P450 enzymes responsible for the oxidative metabolism of the substituted (S)-3-phenylpiperidine, (S,S)-3-[3-(methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride, in human liver microsomes.

(S,S)-3-[3-(Methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride [(-)-OSU6162] is a weak dopamine D2 receptor modulator that possesses potential for the treatment of levodopa (L-DOPA)-induced dyskinesias in patients with Parkinson's disease. In this report, incubations with human liver microsomes revealed that (-)-OSU6162 is selectively metabolized via N-dealkylation to yield N-depropyl (-)-OSU6162. Kinetics evidence is presented that the N-depropylation of (-)-OSU6162 in human hepatic microsomes is mediated by multiple cytochrome p450 (p450) enzymes, in particular CYP2D6. This hypothesis is borne out by several lines of in vitro evidence; 1). incubations of (-)-OSU6162 (5 micro M) with hepatic microsomes from a panel of human donors showed that (-)-OSU6162 N-depropylase activity correlated well with CYP2D6-catalyzed dextromethorphan O-demethylase activity but not with other p450 enzyme-specific activities; 2). quinidine, a CYP2D6-specific inhibitor, inhibited (-)-OSU6162 N-depropylation, whereas other p450 enzyme-specific substrates/inhibitors did not significantly inhibit this activity; 3). CYP2D6 possessed highest intrinsic (-)-OSU6162 N-depropylase activity when compared with a battery of recombinant heterologously expressed human p450 enzymes. In addition, the selectivity of (-)-OSU6162 to inhibit six human p450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6 and CYP3A4) was evaluated using an in vitro inhibition screen. Of the enzymes examined, only the activity of CYP2D6 was inhibited by coincubation with (-)-OSU6162. Thus, it is concluded that (-)-OSU6162 is metabolized by several p450 enzymes and that CYP2D6 accounts for the majority of the observed p450 N-depropylase activity in vitro.     

Effect of high sucrose diet on liver enzyme content and activity and aflatoxin B1-induced mutagenesis

Aflatoxin B1 (AFB1) is a fungal toxin and contaminant that has been implicated in human liver carcinogenesis. In this study we evaluated the effect of a 65% of total calories from sucrose diet (HSD) for 90 days on hepatic cytochrome P450 (CYP450) and glutathione-S-transferase (GST) activity compared to rats maintained on standard lab chow (0% sucrose). There was a statistically significant increase in the number of S. typhimurium His+ revertants (p < 0.001) generated from the incubation of AFB1 with hepatic microsomes from rats fed a HSD. The HSD did not affect the total microsomal CYP450 content nor content of CYP450 1A2, 2B1, 2 isoforms which activate AFB1. Alkoxyresorufin O-dealkylase activity (MROD, PROD) of microsomes from animals fed HSD was decreased by 73% and 49%, respectively. MROD activity is linked to CYP 1A2 activity while PROD is linked to CYP 2B1,2 activity. Although the amount of CYP 3A was significantly decreased in rats fed a HSD, its activity, determined by the presence of the fluorometric metabolite 7-hydroxyquinidine, was unchanged. GST activity was significantly lower in the rats fed HSD.     

Tea consumption modulates hepatic drug metabolizing enzymes in Wistar rats

The antioxidant, antimutagenic and anticarcinogenic activities of green tea and its polyphenols have been reported. As bioactivation of the precarcinogens and detoxification of ultimate carcinogens are mainly carried out by hepatic metabolizing enzymes, we have investigated the modulation of these enzyme activities subsequent to tea consumption in rats. Female Wistar rats were divided into eight groups (n = 5). Six groups were given aqueous solutions (2%, w/v) of six different teas (New Zealand green tea, Australian green tea, Java green tea, Dragon green tea, Gunpowder green tea or English Breakfast black tea) as the sole source of fluid. One group was given a standard green tea extract (0.5%, w/v) while the control group had free access to water. At the end of four-weeks treatment, different cytochrome P450 (CYP) isoform and phase II enzyme activities were determined by incubation of the liver microsomes or cytosols with appropriate substrates. CYP 1A2 activity was markedly increased in all the tea treatment groups (P < 0.05). CYP 1A1 activity was increased significantly in most of the groups except for the Madura, Gunpowder, and Java green tea-treatment groups. Cytosolic glutathione-S-transferase activity was significantly increased (P< 0.05) in the New Zealand, Gunpowder, and Java green tea-treatment groups. The microsomal UDP-glucuronosyl transferase activity remained unchanged or was moderately increased in most of the groups. The balance between the phase I carcinogen-activating enzymes and the phase II detoxifying enzymes could be important in determining the risk of developing chemically-induced cancer.