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Effect of vanillin and ethyl vanillin on cytochrome P450 activity in vitro and in vivo
Food and Chemical Toxicology 50 (2012) 1897–1901
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Effect of vanillin and ethyl vanillin on cytochrome P450 activity in vitro and in vivo Xiao-min Chen b,1, Min Wei a,1, Hai-mou Zhang b, Cheng-hao Luo a, Yi-kun Chen a, Yong Chen b,⇑ a b
Technology Center of China Tobacco Hubei Industry Limited–Liability Company, Wuhan 430040, China Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, Hubei University, Wuhan 430062, China
a r t i c l e
i n f o
Article history: Received 25 November 2011 Accepted 15 March 2012 Available online 23 March 2012 Keywords: Vanillin Ethyl vanillin Cytochrome P450 Liver microsome Enzyme activity
a b s t r a c t Food safety is of extreme importance to human health. Vanillin and ethyl vanillin are the widely used food additives and spices in foods, beverages, cosmetics and drugs. The objective of the present work was to evaluate the impact of vanillin and ethyl vanillin on the activities of CYP2C9, CYP2E1, CYP3A4, CYP2B6 and CYP1A2 in human liver microsomes (HLM) in vitro, and impact on the activities of CYP1A2, CYP2C, CYP3A and CYP2E1 in rat liver microsomes (RLM) in vivo. The in vitro results demonstrated that vanillin and ethyl vanillin had no significant effect on the activity of five human CYP450 enzymes with concentration ranged from 8 to 128 lM. However, after rats were orally administered vanillin or ethyl vanillin once a day for seven consecutive days, CYP2E1 activity was increased and CYP1A2 activity was decreased in RLM. The in vivo results revealed that drug interaction between vanillin/ethyl vanillin and the CYP2E1/CYP1A2-metabolizing drugs might be possible, and also suggested that the application of the above additives in foods and drugs should not be unlimited so as to avoid the adverse interaction. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Cytochrome P450 enzymes (CYPs) comprise a super-gene family and play key roles in the metabolism of drugs, xenobiotics and endogenous compounds in animals and human. Clinically, drug interactions associated with induction or inhibition of CYPs have been shown to be among the important factors in causing side effects in human (Guengerich, 2006). Nowadays, human liver microsomes and recombinant P450 isoforms (CYP2C9, CYP2E1, CYP3A4, CYP2B6, and CYP1A2) are the preferred in vitro test system for predicting drug–drug interactions (DDI) (Bjornsson et al., 2003). Because some of CYPs isoforms (such as CYP1A2, CYP2C, CYP3A and CYP2E1) in rat are similar to that of human, rats are also always used to predict human DDI associated with induction or inhibition of CYPs activities in vivo (Bogaards et al., 2000). Vanillin and ethyl vanillin (Fig. 1a and b) are popularly used as the food additives nowadays. Previous studies showed that vanillin is useful as anti-sickle cell anemia (Zhang et al., 2004), anti-mutagen (Sasaki et al., 1990; Ho et al., 2009) and anti-bacteria agent (Rupasinghe et al., 2005) at high concentration of un-oxidized form to be medically effective, as well as antioxidant (Tai et al., 2011). Recently, Ho et al. (2011) reported that vanillin administration at high concentration (150 and 300 mg/kg) had no obvious effect on the expression of most xenobiotic metabolism, cell progression,
⇑ Corresponding author. Tel./fax: +86 27 88663590. 1
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0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.03.060
tumor suppressor, DNA damage and inflammation genes in rat brain, and postulated that vanillin could provide blood and brain protective properties. So far, little is known about the effect of vanillin and ethyl vanillin on the activities of CYPs in vitro and in vivo. The drug interactions associated with induction or inhibition of CYP enzymes have been shown to be among the important factors in causing side effects in clinics (Guengerich, 1997). The objective of the present work was to evaluate the impact of vanillin and ethyl vanillin on the activities of CYP2C9, CYP2E1, CYP3A4, CYP2B6 and CYP1A2 in HLM in vitro, and the impact on the activities of CYP1A2, CYP2C, CYP3A and CYP2E1 in RLM in vivo.
2. Materials and methods 2.1. Chemicals and reagents Nicotinamide adenine dinucleotide phosphate reduced (NADPH) was purchased from Roche Co. (Basel, Switzerland). Vanillin and ethyl vanillin were purchased from Acros Organics (New Jersey, USA). Resorufin sodium salt, 4-nitrocatechol and p-nitrophenol were purchased from Sigma–Aldrich Chemical Co. (St. Louis, USA). Testosterone and tolbutamide were purchased from Dr. Ehrenstorfer (Ausberg, Germany). Chlorzoxazone was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ethoxyresorufin, nirvanol, 40 -hydroxytolbutamide, (S)-mephenytoin, 6-hydroxychlorzoxazone were purchased from TRC (Toronto, Canada). Phenacetin was purchased from Alfa Aesar (MA, USA). Acetaminophen was purchased from TCI (Tokyo, Japan). 6bHydroxytestosterone was purchased from Caymen (Michigan, USA). Pooled human liver microsomes were purchased from BD Gentest (Woburn, MA, USA). Methanol and acetonitrile (HPLC grade) were purchased from Dikma Company (Beijing, China). All other chemicals and reagents were of analytical grade and were obtained commercially.
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Fig. 1. Structures of vanillin (a) and ethyl vanillin (b).
2.2. In vitro experiments 2.2.1. Incubation in human liver microsomes All incubations were performed at 37 °C in a system containing human liver microsomal protein (0.5 mg/mL for testosterone 6b-hydroxylation, (S)-mephenytoin N-demethylation and chlorzoxazone 6-hydroxylation, 0.75 mg/mL for phenacetin O-deethylation, 1 mg/mL for tolbutamide 40 -hydroxylation) and a NADPH regenerating system (NADPH 1 mM, MgCl2 5 mM) in the final volume of 200 lL (0.1 M potassium phosphate buffer, pH 7.4). After preincubation at 37 °C for 3 min, the incubation reaction was started by the addition of 1 mM NADPH in phosphate buffer. The duration of incubation was 60 min for testosterone and chlorzoxazone, 45 min for phenacetin and tolbutamide, and 30 min for (S)-mephenytoin. The reaction was stopped by cooling on ice and addition of 400 lL methanol (testosterone 6b-hydroxylation, phenacetin O-deethylation, tolbutamide 40 -hydroxylation), 800 lL ethyl acetate (chlorzoxazone 6-hydroxylation), 500 lL methanol ((S)-mephenytoin N-demethylation). Protein was precipitated by vortexing for 3 min and centrifugation at 12, 000g for 10 min, then the supernatant was evaporated (BioTron Ecospin 3180C LABCONCO, Korea) at 40 °C. The residue was stored at 20 °C until HPLC analysis. The probe substrate concentrations were chosen either at or near the apparent Km values of each CYP enzyme (Guengerich, 2006; Bjornsson et al., 2003; Guo and Wang, 2007; Ko et al., 1998). In this work, the probe substrate concentrations of CYP 1A2, 2C9, 2B6, 2E1 and 3A4 (phenacetin, tolbutamide, (S)-mephenytoin, chlorzoxazone and testosterone) were 50, 150, 1000, 75 and 100 lM, respectively. The concentrations of vanillin/ethyl vanillin added to the incubations were ranged from 8 to 128 lM. 2.2.2. Determination of CYPs activities To determine the production of acetaminophen, 40 -hydroxytolbutamide, nirvanol, 6-hydroxychlorzoxazone, and 6b-hydroxytestosterone, the residue was reconstituted with 100 lL of mobile phase and analyzed by previously published HPLC methods with slight modification (Jurica et al., 2010; Relling et al., 1990; Ko et al., 1998; Cao et al., 2008; Qiu et al., 2010). The separation was performed by using a Zorbax Eclipse XDB–C18 column (5 lm, 150 4.6 mm; Agilent, Waldbronn, Germany) fitted with a Zorbax C18 (5 lm, 12.5 4.6 mm; Agilent, Waldbronn, Germany) guard column. All metabolites were delivered at a flow rate of 0.8 mL/min. Other chromatographic conditions were displayed in Table 1. The quality control (QC) samples of each metabolite were prepared at three concentration (low, middle and high) levels in HLM, and were used to evaluate the precision and relative recovery of the analytical methods established in the present work.
Hubei province, Certificate Number: SCXK 2008-0005) of 6-week-old were housed for 7 days under SPF grade laboratory conditions (25 ± 2 °C, 60 ± 5% relative humidity, 12 h light–dark cycle) with free access to food and tap water throughout the experiments. A total of 21 rats were randomly divided into seven groups (n = 3/ group) and receiving an oral dose of A: saline containing 0.5% CMC; B: vanillin 3 mg/kg/day; C: vanillin 15 mg/kg/day; D: vanillin 75 mg/kg/day; E: ethyl vanillin 3 mg/kg/day; F: ethyl vanillin 15 mg/kg/day; G: ethyl vanillin 75 mg/kg/day once a day for seven consecutive days. Rats were killed by cervical fracture and decapitation 1 h after the last treatment and livers were immediately removed and weighed. The minces of livers were washed with cold homogenization buffer (0.25 mol L1sucrose, 0.01 mol L1Tris–HCl, 1 mmol L1 EDTA, pH 7.4) and used to prepare RLM according to the literature (Peng et al., 2009). RLM samples were stored at 80 °C until use. Protein concentration of RLM was determined by BCA Protein Assay Kit (Thermo Scientific, Rockford, IL) standardized with bovine serum albumin. 2.3.2. Incubation in rat liver microsomes All incubations were performed at 37 °C in a system containing rat microsomal protein (1 mg/mL for testosterone 6b-hydroxylation, p-nitrophenol hydroxylation and tolbutamide 40 -hydroxylation, 0.4 mg/mL for ethoxyresorufin O-deethylation) and a NADPH regenerating system (NADPH, 1 mM, MgCl2, 5 mM) in a final volume of 200 lL (0.1 M potassium phosphate buffer, pH 7.4). After preincubation at 37 °C for 3 min, the incubation reaction was started by the addition of 1 mM NADPH in phosphate buffer. The duration of incubation was 10 min for testosterone and chlorzoxazone, 15 min for ethoxyresorufin and 45 min for tolbutamide. The reaction was stopped by cooling on ice and addition of 400 lL methanol (testosterone 6b-hydroxylation, tolbutamide 40 -hydroxylation and ethoxyresorufin O-deethylation) and 100 lL HClO4 (0.6 M) (p-nitrophenol hydroxylation). Protein was precipitated by vortexing for 3 min and centrifugation at 12, 000 g for 10 min, then the supernatants (6b-hydroxytestosterone and 40 -hydroxytolbutamide) were evaporated (BioTron Ecospin 3180C LABCONCO, Korea) at 40 °C. The residues and supernatants were stored at 20 °C until analysis. The substrate concentrations used for CYP 1A2, 2C, 2E1 and 3A (ethoxyresorufin, tolbutamide, p-nitrophenol and testosterone) were 6.5, 250, 500 and 250 lM, respectively. 2.3.3. Determination of CYPs activities The productions of 40 -hydroxytolbutamide, 4-nitrocatechol, resorufin and 6bhydroxytestosterone were determined by previously published methods (Relling et al., 1990; Allis and Robinson, 1994; Sarich et al., 1999; Qiu et al., 2010) with slight modification. HPLC methods for 6b-hydroxytestosterone and 40 -hydroxytolbutamide were the same as that of determinations of CYP3A4 and CYP 2C9 activities in HLM samples. Resorufin was determined by using a Multi-function Microplate Reader (TriStar LB 941, Berthold Technologies, Germany) with the excitation and emission wavelength at 530 and 586 nm. The absorbance of 4-nitrocatechol was determined at 530 nm. 2.4. Statistical analysis Data were expressed as mean ± standard deviation (SD). Two-way analysis of variance (two-way ANOVA) and the Student–Newman–Keuls multiple range test using Sigmastat TM (Jandel Co., SPSS Science, Chicago, IL, USA) were performed.
3. Results
2.3. In vivo experiments 2.3.1. Animal treatments and preparation of liver microsomes According to the acceptable daily intake (ADI) of vanillin and ethyl vanillin (0– 10 and 0–3 mg/kg of body weight for humans, respectively) (JECFA, 2002), the low, medium and high oral dosages of rats were 3, 15 and 75 mg/kg vanillin/ethyl vanillin per day in the present work. All animal procedures were conducted in accordance with guidelines for the care and the use of laboratory animals and approved by the Animal Care Committee of Hubei University. Male Wistar rats (180–200 g, Experimental Animal Center of
3.1. Effect in vitro 3.1.1. Method validation The retention time of acetaminophen, nirvanol, 40 -hydroxytolbutamide, 6-hydroxychlorzoxazone and 6b-hydroxytestosterone were 3.9 min, 13.5 min, 6.2 min, 5.1 min and 7.8 min, respectively. No significant interference was observed in the controls.
Table 1 HPLC methods for simultaneous determination of five cytochrome P450 (CYP) probe substrate metabolites. CYPs
Metabolites
Mobile phase
UV wavelength (nm)
CYP1A2 CYP2C9 CYP2B6
Acetaminophen 40 -Hydroxytolbutamide Nirvanol
245 230 204
CYP2E1 CYP3A4
6-Hydroxychlorzoxazone 6b-Hydroxytestosterone
23% methanol, 77% water (containing 10 mM potassium dihydrogen phosphate, pH = 4.5) 30% acetonitrile, 70% water (containing 0.1% acetic acid) 11.2% acetonitrile, 16.8% methanol, 72% water (containing 50 mM potassium dihydrogen phosphate, pH = 4.2) 22% acetonitrile, 78% water 55% methanol, 45% water
282 247
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Fig. 2. Effect of vanillin on CYP activity in HLM in vitro. All values are presented as the mean ± SD of triplicate determinations. MCR stands for metabolic clearance rate.
Fig. 3. Effect of ethyl vanillin on CYP activity in HLM in vitro. All values are presented as the mean ± SD of triplicate determinations. MCR stands for metabolic clearance rate.
The linear range of 6b-hydroxytestosterone, acetaminophen, nirvanol, 40 -hydroxytolbutamide and 6-hydroxychlorzoxazone were all ranged from 0.5 to 16.0 lM in HLM mixture without NADPH. Typical equations of calibration curves determined by weighted (1/x2) least squares linear regression are acetaminophen, y = 75.677x + 13.831, (r = 1); 6-hydroxychlorzoxazone, y = 26.746x 0.9541, (r = 0.9999); 40 -hydroxytolbutamide, y = 94.383x + 3.4937, (r = 0.9998); nirvanol, y = 84.427x + 4.5529, (r = 0.9998); 6b-hydroxytestosterone, y = 70.123x 2.267, (r = 0.9998). All calibration curves showed excellent linearity in the concentration ranges used. The mean withinand between-day coefficients of variation for the assay precision were less than 15% and recoveries were precise and reproducible.
3.1.2. Effect of vanillin and ethyl vanillin on five CYPs activities The in vitro effect of vanillin and ethyl vanillin on five CYPs activities in HLM were shown in Figs. 2 and 3. The results indicated that both vanillin and ethyl vanillin had no significant effect on the five CYP-catalyzed reactions in HLM. 3.2. Effect in vivo 3.2.1. Method validation The retention time of 40 -hydroxytolbutamide and 6b-hydroxytestosterone were 5.0 min and 7.9 min, respectively. In addition, the fluorescence intensity of resorufin and the absorbance of
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Table 2 Effect of vanillin on CYP activity in RLM in vivo.
a b c
Activitya/pmol mg1 min1
CYPs
Control
Vanillin (3 mg kg1 d1)
Vanillin (15 mg kg1 d1)
Vanillin (75 mg kg1 d1)
6bTesto–OH PNP–OH EROD Tol–OH
CYP3A CYP2E1 CYP1A2 CYP2C
75.1 ± 23.4 101.3 ± 6.3 8.5 ± 0.3 22.5 ± 1.4
78.5 ± 4.0 165.8 ± 3.6c 6.8 ± 0.2c 23.3 ± 5.4
94.2 ± 10.4 120.0 ± 28.6 7.6 ± 0.7 21.3 ± 5.8
89.7 ± 14.5 136.7 ± 14.4b 6.6 ± 0.6c 22.0 ± 2.2
Data are presented as the mean ± SD of triplicate determinations. Significantly different from the control by two-way analysis of variance and by the Student–Newman–Keuls multiple range test (P < 0.05). Significantly different from the control by two-way analysis of variance and by the Student–Newman–Keuls multiple range test (P < 0.01).
Table 3 Effect of ethyl vanillin on CYP activity in RLM in vivo.
a b c
Activitya (pmol mg1 min1)
CYPs
Control
Ethyl vanillin (3 mg kg1 d1)
Ethyl vanillin (15 mg kg1 d1)
Ethyl vanillin (75 mg kg1 d1)
6bTesto–OH PNP–OH EROD Tol–OH
CYP3A CYP2E1 CYP1A2 CYP2C
75.1 ± 23.4 101.3 ± 6.3 8.5 ± 0.3 22.5 ± 1.4
72.5 ± 1.5 117.9 ± 7.2b 7.0 ± 0.7b 20.7 ± 4.5
62.2 ± 3.2 120.0 ± 33.1 5.9 ± 0.3c 20.0 ± 4.6
90.6 ± 14.2 132.5 ± 6.3c 6.9 ± 1.3 21.6 ± 6.3
Data are presented as the mean ± SD of triplicate determinations. Significantly different from the control by two-way analysis of variance and by the Student–Newman–Keuls multiple range test (P < 0.05). Significantly different from the control by two-way analysis of variance and by the Student–Newman–Keuls multiple range test (P < 0.01).
4-nitrocatechol in blank RLM samples are all significantly lower than that of their samples at LOQs. The results indicated that no significant interference was observed in the controls. The linearity were ranged from 0.5 to 16.0 lM for 6b-hydroxytestosterone (6bTesto–OH), 0.25 to 8.0 lM for 40 -hydroxytolbutamide (Tol–OH), 0.5 to 80.0 lM for 4-nitrocatechol (PNP–OH), 12.5 to 800 nM for resorufin (EROD) in RLM mixture without NADPH. Typical equations of calibration curves determined by weighted (1/x2) least squares linear regression are resorufin, y = 16.928x + 65.816, (r = 0.9999); 4-nitrocatechol, y = 0.0032x 0.0002, (r = 0.9998); 40 hydroxytolbutamide, y = 70.189x + 46.774, (r = 0.9996); 6b-hydroxytestosterone, y = 46.963x + 8.0382, (r = 0.9997). All calibration curves showed excellent linearity in the concentration ranges used. The mean within- and between-day coefficients of variation for the assay precision were less than 15% and recoveries were precise and reproducible. 3.2.2. Effect of vanillin and ethyl vanillin on four CYPs activities The in vivo effects of vanillin and ethyl vanillin on four CYPs activities in RLM were shown in Tables 2 and 3. Compared to the control, vanillin at low, medium and high doses caused 19.4%, 9.8% and 22.6% decreases in CYP1A2-associated resorufin formation respectively, and caused 63.7%, 18.5% and 34.9% increases in CYP2E1-associated p-nitrocatechol formation respectively, but the change caused by medium dose had no significant difference. Ethyl vanillin at low, medium and high doses caused 17.2%, 30.2% and 19.0% decreases in CYP1A2-associated resorufin formation respectively, and caused 16.4%, 18.5% and 30.8% increases in CYP2E1-associated p-nitrocatechol formation respectively. But the change of CYP1A2 activity caused by high dose, and the change of CYP2E1 activity caused by medium dose, had no significant difference. In addition, Vanillin and ethyl vanillin had no significant effect on the activities of CYP3A and CYP2C in RLM. The results indicated that both vanillin and ethyl vanillin had an inhibition for CYP1A2 activity and an induction for CYP2E1 activity in some extent in vivo. 4. Discussion Food safety is of extreme importance to human health. Food additives play an important role in maintaining the food qualities and characteristics, and keeping food safe, wholesome and appealing. Vanillin and ethyl vanillin are the widely used food additives
and spices in food, beverage, cosmetic and drug. In the present work, the in vitro results demonstrated that vanillin and ethyl vanillin had no significant effect on the activities of five human P450 enzymes (CYP1A2, 3A4, 2B6, 2C9, 2E1) with the concentration ranged from 8 to 128 lM. However, after rats were orally administered vanillin or ethyl vanillin once a day for seven consecutive days, the induction for CYP2E1 and the inhibition for CYP1A2 in RLM revealed that the DDI between both of the food additives and CYP2E1/CYP1A2-metabolizing drugs might be possible. It is well known that the metabolic activation of carcinogens is mainly mediated by CYP2E1 and CYP1A1/2. The decrease in both CYP2E1 and CYP1A1/2 activities can obviously inhibit the biotransformation of some chemicals in liver, and then attenuate their carcinogenicity and chemical-induced hepatic injury (Gonzalez, 2005; Gunes and Dahl, 2008; Yao et al., 2009). Therefore, the in vivo results also suggested that the drug interactions between vanillin/ethyl vanillin and the CYP2E1/CYP1A2-metabolizing drugs might be possible, and the application of the above additives should not be unlimited so as to avoid the adverse interaction due to the change of CYP2E1 and CYP1A2 activities. 5. Conclusion Vanillin and ethyl vanillin are widely used as food additives nowadays. Results of this study showed that both vanillin and ethyl vanillin had no significant effect on the five CYP-catalyzed reactions in vitro. However, the induction for CYP2E1 and the inhibition for CYP1A2 in RLM in vivo caused by oral administration of vanillin and ethyl vanillin revealed that the DDI between both of the food additives and CYP2E1/CYP1A2-metabolizing drugs might be possible. We indicated that the application of these two additives in food industry should be more careful so as to avoid the adverse interaction. Conflict of Interest The authors declare that there are no conflicts of interest. References Allis, J.W., Robinson, B.L., 1994. A kinetic assay for p-nitrophenol hydroxylase in rat liver microsomes. Anal. Biochem. 219, 49–52.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Effect of vanillin and ethyl vanillin on cytochrome P450 activity in vitro and in vivo Xiao-min Chen b,1, Min Wei a,1, Hai-mou Zhang b, Cheng-hao Luo a, Yi-kun Chen a, Yong Chen b,⇑ a b
Technology Center of China Tobacco Hubei Industry Limited–Liability Company, Wuhan 430040, China Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, Hubei University, Wuhan 430062, China
a r t i c l e
i n f o
Article history: Received 25 November 2011 Accepted 15 March 2012 Available online 23 March 2012 Keywords: Vanillin Ethyl vanillin Cytochrome P450 Liver microsome Enzyme activity
a b s t r a c t Food safety is of extreme importance to human health. Vanillin and ethyl vanillin are the widely used food additives and spices in foods, beverages, cosmetics and drugs. The objective of the present work was to evaluate the impact of vanillin and ethyl vanillin on the activities of CYP2C9, CYP2E1, CYP3A4, CYP2B6 and CYP1A2 in human liver microsomes (HLM) in vitro, and impact on the activities of CYP1A2, CYP2C, CYP3A and CYP2E1 in rat liver microsomes (RLM) in vivo. The in vitro results demonstrated that vanillin and ethyl vanillin had no significant effect on the activity of five human CYP450 enzymes with concentration ranged from 8 to 128 lM. However, after rats were orally administered vanillin or ethyl vanillin once a day for seven consecutive days, CYP2E1 activity was increased and CYP1A2 activity was decreased in RLM. The in vivo results revealed that drug interaction between vanillin/ethyl vanillin and the CYP2E1/CYP1A2-metabolizing drugs might be possible, and also suggested that the application of the above additives in foods and drugs should not be unlimited so as to avoid the adverse interaction. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Cytochrome P450 enzymes (CYPs) comprise a super-gene family and play key roles in the metabolism of drugs, xenobiotics and endogenous compounds in animals and human. Clinically, drug interactions associated with induction or inhibition of CYPs have been shown to be among the important factors in causing side effects in human (Guengerich, 2006). Nowadays, human liver microsomes and recombinant P450 isoforms (CYP2C9, CYP2E1, CYP3A4, CYP2B6, and CYP1A2) are the preferred in vitro test system for predicting drug–drug interactions (DDI) (Bjornsson et al., 2003). Because some of CYPs isoforms (such as CYP1A2, CYP2C, CYP3A and CYP2E1) in rat are similar to that of human, rats are also always used to predict human DDI associated with induction or inhibition of CYPs activities in vivo (Bogaards et al., 2000). Vanillin and ethyl vanillin (Fig. 1a and b) are popularly used as the food additives nowadays. Previous studies showed that vanillin is useful as anti-sickle cell anemia (Zhang et al., 2004), anti-mutagen (Sasaki et al., 1990; Ho et al., 2009) and anti-bacteria agent (Rupasinghe et al., 2005) at high concentration of un-oxidized form to be medically effective, as well as antioxidant (Tai et al., 2011). Recently, Ho et al. (2011) reported that vanillin administration at high concentration (150 and 300 mg/kg) had no obvious effect on the expression of most xenobiotic metabolism, cell progression,
⇑ Corresponding author. Tel./fax: +86 27 88663590. 1
E-mail address: [email protected] (Y. Chen). Both the authors contributed equally and both are co-first authors.
0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.03.060
tumor suppressor, DNA damage and inflammation genes in rat brain, and postulated that vanillin could provide blood and brain protective properties. So far, little is known about the effect of vanillin and ethyl vanillin on the activities of CYPs in vitro and in vivo. The drug interactions associated with induction or inhibition of CYP enzymes have been shown to be among the important factors in causing side effects in clinics (Guengerich, 1997). The objective of the present work was to evaluate the impact of vanillin and ethyl vanillin on the activities of CYP2C9, CYP2E1, CYP3A4, CYP2B6 and CYP1A2 in HLM in vitro, and the impact on the activities of CYP1A2, CYP2C, CYP3A and CYP2E1 in RLM in vivo.
2. Materials and methods 2.1. Chemicals and reagents Nicotinamide adenine dinucleotide phosphate reduced (NADPH) was purchased from Roche Co. (Basel, Switzerland). Vanillin and ethyl vanillin were purchased from Acros Organics (New Jersey, USA). Resorufin sodium salt, 4-nitrocatechol and p-nitrophenol were purchased from Sigma–Aldrich Chemical Co. (St. Louis, USA). Testosterone and tolbutamide were purchased from Dr. Ehrenstorfer (Ausberg, Germany). Chlorzoxazone was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ethoxyresorufin, nirvanol, 40 -hydroxytolbutamide, (S)-mephenytoin, 6-hydroxychlorzoxazone were purchased from TRC (Toronto, Canada). Phenacetin was purchased from Alfa Aesar (MA, USA). Acetaminophen was purchased from TCI (Tokyo, Japan). 6bHydroxytestosterone was purchased from Caymen (Michigan, USA). Pooled human liver microsomes were purchased from BD Gentest (Woburn, MA, USA). Methanol and acetonitrile (HPLC grade) were purchased from Dikma Company (Beijing, China). All other chemicals and reagents were of analytical grade and were obtained commercially.
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Fig. 1. Structures of vanillin (a) and ethyl vanillin (b).
2.2. In vitro experiments 2.2.1. Incubation in human liver microsomes All incubations were performed at 37 °C in a system containing human liver microsomal protein (0.5 mg/mL for testosterone 6b-hydroxylation, (S)-mephenytoin N-demethylation and chlorzoxazone 6-hydroxylation, 0.75 mg/mL for phenacetin O-deethylation, 1 mg/mL for tolbutamide 40 -hydroxylation) and a NADPH regenerating system (NADPH 1 mM, MgCl2 5 mM) in the final volume of 200 lL (0.1 M potassium phosphate buffer, pH 7.4). After preincubation at 37 °C for 3 min, the incubation reaction was started by the addition of 1 mM NADPH in phosphate buffer. The duration of incubation was 60 min for testosterone and chlorzoxazone, 45 min for phenacetin and tolbutamide, and 30 min for (S)-mephenytoin. The reaction was stopped by cooling on ice and addition of 400 lL methanol (testosterone 6b-hydroxylation, phenacetin O-deethylation, tolbutamide 40 -hydroxylation), 800 lL ethyl acetate (chlorzoxazone 6-hydroxylation), 500 lL methanol ((S)-mephenytoin N-demethylation). Protein was precipitated by vortexing for 3 min and centrifugation at 12, 000g for 10 min, then the supernatant was evaporated (BioTron Ecospin 3180C LABCONCO, Korea) at 40 °C. The residue was stored at 20 °C until HPLC analysis. The probe substrate concentrations were chosen either at or near the apparent Km values of each CYP enzyme (Guengerich, 2006; Bjornsson et al., 2003; Guo and Wang, 2007; Ko et al., 1998). In this work, the probe substrate concentrations of CYP 1A2, 2C9, 2B6, 2E1 and 3A4 (phenacetin, tolbutamide, (S)-mephenytoin, chlorzoxazone and testosterone) were 50, 150, 1000, 75 and 100 lM, respectively. The concentrations of vanillin/ethyl vanillin added to the incubations were ranged from 8 to 128 lM. 2.2.2. Determination of CYPs activities To determine the production of acetaminophen, 40 -hydroxytolbutamide, nirvanol, 6-hydroxychlorzoxazone, and 6b-hydroxytestosterone, the residue was reconstituted with 100 lL of mobile phase and analyzed by previously published HPLC methods with slight modification (Jurica et al., 2010; Relling et al., 1990; Ko et al., 1998; Cao et al., 2008; Qiu et al., 2010). The separation was performed by using a Zorbax Eclipse XDB–C18 column (5 lm, 150 4.6 mm; Agilent, Waldbronn, Germany) fitted with a Zorbax C18 (5 lm, 12.5 4.6 mm; Agilent, Waldbronn, Germany) guard column. All metabolites were delivered at a flow rate of 0.8 mL/min. Other chromatographic conditions were displayed in Table 1. The quality control (QC) samples of each metabolite were prepared at three concentration (low, middle and high) levels in HLM, and were used to evaluate the precision and relative recovery of the analytical methods established in the present work.
Hubei province, Certificate Number: SCXK 2008-0005) of 6-week-old were housed for 7 days under SPF grade laboratory conditions (25 ± 2 °C, 60 ± 5% relative humidity, 12 h light–dark cycle) with free access to food and tap water throughout the experiments. A total of 21 rats were randomly divided into seven groups (n = 3/ group) and receiving an oral dose of A: saline containing 0.5% CMC; B: vanillin 3 mg/kg/day; C: vanillin 15 mg/kg/day; D: vanillin 75 mg/kg/day; E: ethyl vanillin 3 mg/kg/day; F: ethyl vanillin 15 mg/kg/day; G: ethyl vanillin 75 mg/kg/day once a day for seven consecutive days. Rats were killed by cervical fracture and decapitation 1 h after the last treatment and livers were immediately removed and weighed. The minces of livers were washed with cold homogenization buffer (0.25 mol L1sucrose, 0.01 mol L1Tris–HCl, 1 mmol L1 EDTA, pH 7.4) and used to prepare RLM according to the literature (Peng et al., 2009). RLM samples were stored at 80 °C until use. Protein concentration of RLM was determined by BCA Protein Assay Kit (Thermo Scientific, Rockford, IL) standardized with bovine serum albumin. 2.3.2. Incubation in rat liver microsomes All incubations were performed at 37 °C in a system containing rat microsomal protein (1 mg/mL for testosterone 6b-hydroxylation, p-nitrophenol hydroxylation and tolbutamide 40 -hydroxylation, 0.4 mg/mL for ethoxyresorufin O-deethylation) and a NADPH regenerating system (NADPH, 1 mM, MgCl2, 5 mM) in a final volume of 200 lL (0.1 M potassium phosphate buffer, pH 7.4). After preincubation at 37 °C for 3 min, the incubation reaction was started by the addition of 1 mM NADPH in phosphate buffer. The duration of incubation was 10 min for testosterone and chlorzoxazone, 15 min for ethoxyresorufin and 45 min for tolbutamide. The reaction was stopped by cooling on ice and addition of 400 lL methanol (testosterone 6b-hydroxylation, tolbutamide 40 -hydroxylation and ethoxyresorufin O-deethylation) and 100 lL HClO4 (0.6 M) (p-nitrophenol hydroxylation). Protein was precipitated by vortexing for 3 min and centrifugation at 12, 000 g for 10 min, then the supernatants (6b-hydroxytestosterone and 40 -hydroxytolbutamide) were evaporated (BioTron Ecospin 3180C LABCONCO, Korea) at 40 °C. The residues and supernatants were stored at 20 °C until analysis. The substrate concentrations used for CYP 1A2, 2C, 2E1 and 3A (ethoxyresorufin, tolbutamide, p-nitrophenol and testosterone) were 6.5, 250, 500 and 250 lM, respectively. 2.3.3. Determination of CYPs activities The productions of 40 -hydroxytolbutamide, 4-nitrocatechol, resorufin and 6bhydroxytestosterone were determined by previously published methods (Relling et al., 1990; Allis and Robinson, 1994; Sarich et al., 1999; Qiu et al., 2010) with slight modification. HPLC methods for 6b-hydroxytestosterone and 40 -hydroxytolbutamide were the same as that of determinations of CYP3A4 and CYP 2C9 activities in HLM samples. Resorufin was determined by using a Multi-function Microplate Reader (TriStar LB 941, Berthold Technologies, Germany) with the excitation and emission wavelength at 530 and 586 nm. The absorbance of 4-nitrocatechol was determined at 530 nm. 2.4. Statistical analysis Data were expressed as mean ± standard deviation (SD). Two-way analysis of variance (two-way ANOVA) and the Student–Newman–Keuls multiple range test using Sigmastat TM (Jandel Co., SPSS Science, Chicago, IL, USA) were performed.
3. Results
2.3. In vivo experiments 2.3.1. Animal treatments and preparation of liver microsomes According to the acceptable daily intake (ADI) of vanillin and ethyl vanillin (0– 10 and 0–3 mg/kg of body weight for humans, respectively) (JECFA, 2002), the low, medium and high oral dosages of rats were 3, 15 and 75 mg/kg vanillin/ethyl vanillin per day in the present work. All animal procedures were conducted in accordance with guidelines for the care and the use of laboratory animals and approved by the Animal Care Committee of Hubei University. Male Wistar rats (180–200 g, Experimental Animal Center of
3.1. Effect in vitro 3.1.1. Method validation The retention time of acetaminophen, nirvanol, 40 -hydroxytolbutamide, 6-hydroxychlorzoxazone and 6b-hydroxytestosterone were 3.9 min, 13.5 min, 6.2 min, 5.1 min and 7.8 min, respectively. No significant interference was observed in the controls.
Table 1 HPLC methods for simultaneous determination of five cytochrome P450 (CYP) probe substrate metabolites. CYPs
Metabolites
Mobile phase
UV wavelength (nm)
CYP1A2 CYP2C9 CYP2B6
Acetaminophen 40 -Hydroxytolbutamide Nirvanol
245 230 204
CYP2E1 CYP3A4
6-Hydroxychlorzoxazone 6b-Hydroxytestosterone
23% methanol, 77% water (containing 10 mM potassium dihydrogen phosphate, pH = 4.5) 30% acetonitrile, 70% water (containing 0.1% acetic acid) 11.2% acetonitrile, 16.8% methanol, 72% water (containing 50 mM potassium dihydrogen phosphate, pH = 4.2) 22% acetonitrile, 78% water 55% methanol, 45% water
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Fig. 2. Effect of vanillin on CYP activity in HLM in vitro. All values are presented as the mean ± SD of triplicate determinations. MCR stands for metabolic clearance rate.
Fig. 3. Effect of ethyl vanillin on CYP activity in HLM in vitro. All values are presented as the mean ± SD of triplicate determinations. MCR stands for metabolic clearance rate.
The linear range of 6b-hydroxytestosterone, acetaminophen, nirvanol, 40 -hydroxytolbutamide and 6-hydroxychlorzoxazone were all ranged from 0.5 to 16.0 lM in HLM mixture without NADPH. Typical equations of calibration curves determined by weighted (1/x2) least squares linear regression are acetaminophen, y = 75.677x + 13.831, (r = 1); 6-hydroxychlorzoxazone, y = 26.746x 0.9541, (r = 0.9999); 40 -hydroxytolbutamide, y = 94.383x + 3.4937, (r = 0.9998); nirvanol, y = 84.427x + 4.5529, (r = 0.9998); 6b-hydroxytestosterone, y = 70.123x 2.267, (r = 0.9998). All calibration curves showed excellent linearity in the concentration ranges used. The mean withinand between-day coefficients of variation for the assay precision were less than 15% and recoveries were precise and reproducible.
3.1.2. Effect of vanillin and ethyl vanillin on five CYPs activities The in vitro effect of vanillin and ethyl vanillin on five CYPs activities in HLM were shown in Figs. 2 and 3. The results indicated that both vanillin and ethyl vanillin had no significant effect on the five CYP-catalyzed reactions in HLM. 3.2. Effect in vivo 3.2.1. Method validation The retention time of 40 -hydroxytolbutamide and 6b-hydroxytestosterone were 5.0 min and 7.9 min, respectively. In addition, the fluorescence intensity of resorufin and the absorbance of
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Table 2 Effect of vanillin on CYP activity in RLM in vivo.
a b c
Activitya/pmol mg1 min1
CYPs
Control
Vanillin (3 mg kg1 d1)
Vanillin (15 mg kg1 d1)
Vanillin (75 mg kg1 d1)
6bTesto–OH PNP–OH EROD Tol–OH
CYP3A CYP2E1 CYP1A2 CYP2C
75.1 ± 23.4 101.3 ± 6.3 8.5 ± 0.3 22.5 ± 1.4
78.5 ± 4.0 165.8 ± 3.6c 6.8 ± 0.2c 23.3 ± 5.4
94.2 ± 10.4 120.0 ± 28.6 7.6 ± 0.7 21.3 ± 5.8
89.7 ± 14.5 136.7 ± 14.4b 6.6 ± 0.6c 22.0 ± 2.2
Data are presented as the mean ± SD of triplicate determinations. Significantly different from the control by two-way analysis of variance and by the Student–Newman–Keuls multiple range test (P < 0.05). Significantly different from the control by two-way analysis of variance and by the Student–Newman–Keuls multiple range test (P < 0.01).
Table 3 Effect of ethyl vanillin on CYP activity in RLM in vivo.
a b c
Activitya (pmol mg1 min1)
CYPs
Control
Ethyl vanillin (3 mg kg1 d1)
Ethyl vanillin (15 mg kg1 d1)
Ethyl vanillin (75 mg kg1 d1)
6bTesto–OH PNP–OH EROD Tol–OH
CYP3A CYP2E1 CYP1A2 CYP2C
75.1 ± 23.4 101.3 ± 6.3 8.5 ± 0.3 22.5 ± 1.4
72.5 ± 1.5 117.9 ± 7.2b 7.0 ± 0.7b 20.7 ± 4.5
62.2 ± 3.2 120.0 ± 33.1 5.9 ± 0.3c 20.0 ± 4.6
90.6 ± 14.2 132.5 ± 6.3c 6.9 ± 1.3 21.6 ± 6.3
Data are presented as the mean ± SD of triplicate determinations. Significantly different from the control by two-way analysis of variance and by the Student–Newman–Keuls multiple range test (P < 0.05). Significantly different from the control by two-way analysis of variance and by the Student–Newman–Keuls multiple range test (P < 0.01).
4-nitrocatechol in blank RLM samples are all significantly lower than that of their samples at LOQs. The results indicated that no significant interference was observed in the controls. The linearity were ranged from 0.5 to 16.0 lM for 6b-hydroxytestosterone (6bTesto–OH), 0.25 to 8.0 lM for 40 -hydroxytolbutamide (Tol–OH), 0.5 to 80.0 lM for 4-nitrocatechol (PNP–OH), 12.5 to 800 nM for resorufin (EROD) in RLM mixture without NADPH. Typical equations of calibration curves determined by weighted (1/x2) least squares linear regression are resorufin, y = 16.928x + 65.816, (r = 0.9999); 4-nitrocatechol, y = 0.0032x 0.0002, (r = 0.9998); 40 hydroxytolbutamide, y = 70.189x + 46.774, (r = 0.9996); 6b-hydroxytestosterone, y = 46.963x + 8.0382, (r = 0.9997). All calibration curves showed excellent linearity in the concentration ranges used. The mean within- and between-day coefficients of variation for the assay precision were less than 15% and recoveries were precise and reproducible. 3.2.2. Effect of vanillin and ethyl vanillin on four CYPs activities The in vivo effects of vanillin and ethyl vanillin on four CYPs activities in RLM were shown in Tables 2 and 3. Compared to the control, vanillin at low, medium and high doses caused 19.4%, 9.8% and 22.6% decreases in CYP1A2-associated resorufin formation respectively, and caused 63.7%, 18.5% and 34.9% increases in CYP2E1-associated p-nitrocatechol formation respectively, but the change caused by medium dose had no significant difference. Ethyl vanillin at low, medium and high doses caused 17.2%, 30.2% and 19.0% decreases in CYP1A2-associated resorufin formation respectively, and caused 16.4%, 18.5% and 30.8% increases in CYP2E1-associated p-nitrocatechol formation respectively. But the change of CYP1A2 activity caused by high dose, and the change of CYP2E1 activity caused by medium dose, had no significant difference. In addition, Vanillin and ethyl vanillin had no significant effect on the activities of CYP3A and CYP2C in RLM. The results indicated that both vanillin and ethyl vanillin had an inhibition for CYP1A2 activity and an induction for CYP2E1 activity in some extent in vivo. 4. Discussion Food safety is of extreme importance to human health. Food additives play an important role in maintaining the food qualities and characteristics, and keeping food safe, wholesome and appealing. Vanillin and ethyl vanillin are the widely used food additives
and spices in food, beverage, cosmetic and drug. In the present work, the in vitro results demonstrated that vanillin and ethyl vanillin had no significant effect on the activities of five human P450 enzymes (CYP1A2, 3A4, 2B6, 2C9, 2E1) with the concentration ranged from 8 to 128 lM. However, after rats were orally administered vanillin or ethyl vanillin once a day for seven consecutive days, the induction for CYP2E1 and the inhibition for CYP1A2 in RLM revealed that the DDI between both of the food additives and CYP2E1/CYP1A2-metabolizing drugs might be possible. It is well known that the metabolic activation of carcinogens is mainly mediated by CYP2E1 and CYP1A1/2. The decrease in both CYP2E1 and CYP1A1/2 activities can obviously inhibit the biotransformation of some chemicals in liver, and then attenuate their carcinogenicity and chemical-induced hepatic injury (Gonzalez, 2005; Gunes and Dahl, 2008; Yao et al., 2009). Therefore, the in vivo results also suggested that the drug interactions between vanillin/ethyl vanillin and the CYP2E1/CYP1A2-metabolizing drugs might be possible, and the application of the above additives should not be unlimited so as to avoid the adverse interaction due to the change of CYP2E1 and CYP1A2 activities. 5. Conclusion Vanillin and ethyl vanillin are widely used as food additives nowadays. Results of this study showed that both vanillin and ethyl vanillin had no significant effect on the five CYP-catalyzed reactions in vitro. However, the induction for CYP2E1 and the inhibition for CYP1A2 in RLM in vivo caused by oral administration of vanillin and ethyl vanillin revealed that the DDI between both of the food additives and CYP2E1/CYP1A2-metabolizing drugs might be possible. We indicated that the application of these two additives in food industry should be more careful so as to avoid the adverse interaction. Conflict of Interest The authors declare that there are no conflicts of interest. References Allis, J.W., Robinson, B.L., 1994. A kinetic assay for p-nitrophenol hydroxylase in rat liver microsomes. Anal. Biochem. 219, 49–52.
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