British Journal of Nutrition (2014), 112, 295–308 Doi:10.1017/S0007114514000841 Q the Author 2014

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British Journal of Nutrition (2014), 112, 295–308 doi:10.1017/S0007114514000841 q The Author 2014

https://www.cambridge.org/core

Effect of dietary a-lipoic acid on the mRNA expression of genes involved in drug metabolism and antioxidation system in rat liver

Takashi Ide*† . IP address: Laboratory of Nutritional Function, National Food Research Institute, 2-1-12 Kannondai, Tsukuba 305-8642, Japan

(Submitted 30 July 2013 – Final revision received 11 March 2014 – Accepted 20 March 2014 – First published online 1 May 2014) 170.106.202.226

Abstract In the present study, the mRNA levels of hepatic proteins involved in the drug metabolism of rats fed a-lipoic acid were evaluated by DNA , on microarray and real-time PCR analyses. Experimental diets containing 0, 0·1, 0·25 and 0·5 % (w/w) a-lipoic acid were fed to four groups of 28 Sep 2021 at 21:50:59 rats consisting of seven animals each for 21 d. DNA microarray analysis revealed that the diet containing 0·5 % a-lipoic acid significantly (P,0·05) increased the mRNA levels of various phase I drug-metabolising enzymes up to 15-fold and phase II enzymes up to 52-fold in an isoenzyme-specific manner. a-Lipoic acid also up-regulated the mRNA levels of some members of the ATP-binding cassette transporter superfamily, presumed to be involved in the exportation of xenobiotics, up to 6·6-fold. In addition, we observed that a-lipoic acid increased the mRNA levels of many proteins involved in antioxidation, such as members of the thiol redox system (up to 5·5-fold), metallothioneins (up to 12-fold) and haeme oxygenase 1 (1·5-fold). These results were confirmed using real-time PCR analysis, and a-lipoic acid , subject to the Cambridge Core terms of use, available at dose dependently increased the mRNA levels of various proteins involved in drug metabolism and antioxidation. Consistent with these observations, a-lipoic acid dose dependently increased the hepatic concentration of glutathione and the activities of glutathione reductase and glutathione transferase measured using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, but decreased the hepatic and serum concentrations of malondialdehyde. In conclusion, the present study unequivocally demonstrated that a-lipoic acid increases the mRNA expression of proteins involved in drug metabolism and antioxidation in the liver.

Key words: a-Lipoic acid: DNA microarrays: Drug metabolism: Antioxidation

a-Lipoic acid is a natural compound that is widely distributed at ameliorating insulin resistance and attenuating type 2 in plants and animals and functions as a cofactor within mito- diabetes(1,6,7). The lipid-lowering property of a-lipoic acid has British Journal ofchondrial Nutrition enzymes to catalyse the oxidative decarboxylation also frequently been reported(2,9 – 11). In this context, we had a a a

of -keto acids (such as pyruvate dehydrogenase and -keto- previously demonstrated that -lipoic acid strongly reduces https://www.cambridge.org/core/terms glutarate dehydrogenase) and the cleavage of glycine(1,2). the activity and mRNA levels of hepatic lipogenic enzymes Besides its role in the mitochondrial metabolic pathway, in rats(12 – 14). Therefore, the reduction in hepatic lipid biosyn- a-lipoic acid, when supplemented in diets, performs various thesis may account for the lipid-lowering effect of a-lipoic physiological activities in experimental animals(1,2) and is acid. During the course of the studies, we had found that diet- widely used as a dietary supplement by humans. However, ary a-lipoic acid lowers serum concentrations of TAG, choles- the concentration of a-lipoic acid in natural food items is terol and phospholipids and hepatic concentrations of TAG very low(3). Therefore, dietary a-lipoic acid supplements are and cholesterol, but increases the hepatic concentration of manufactured by chemical synthesis. It has been well estab- phospholipids. The increased hepatic concentration of phos- lished that dietary a-lipoic acid is effective at attenuating pholipids implies that a-lipoic acid causes biomembrane pro- (1,2,4 – 8) a .

oxidative stress . Many studies have also indicated that liferation in this tissue. We hypothesise that -lipoic acid is an https://doi.org/10.1017/S0007114514000841 a-lipoic acid modulates glucose metabolism and is effective inducer of hepatic drug-metabolising enzymes because drugs

Abbreviations: Abc, ATP-binding cassette; Akr, aldo-keto reductase; Aldh, aldehyde dehydrogenase; Apex1, APEX nuclease 1; Ces2c, carboxylesterase 2C; Cyp, cytochrome P450; Ephx1, epoxide hydrolase 1, microsomal; Fmo, flavin-containing monooxygenase; Gclc, glutamate–cysteine ligase, catalytic subunit; Gclm, glutamate–cysteine ligase, modifier subunit; Glrx, glutaredoxin; Gpx, glutathione peroxidase; Gsr, glutathione reductase; Gst, glutathione transferase; Hmox, haeme oxygenase; Mt, metallothionein; NOAEL, no observed adverse effect level; Nqo1, NAD(P)H dehydrogenase, quinone 1; Nr1i3, nuclear receptor subfamily 1, group I, member 3; Nrf2, NFE2-related factor 2; Srxn1, sulfiredoxin 1; Sult, sulfotransferase family, cytosolic; Txnrd1, thioredoxin reductase 1; Ugt, UDP-glucuronosyltransferase. * Corresponding author: T. Ide, fax þ81 48 478 9367, email [email protected]

† Present address: Department of Food and Nutrition, Faculty of Human Life, Jumonji University, 2-1-28 Sugasawa, Niiza, Saitama 352-8510, Japan. Downloaded from

296 T. Ide

that induce hepatic drug metabolism also increase phospho- 54·1 % (w/w) of R-isomers and S-isomers, respectively. https://www.cambridge.org/core lipid concentration and synthesis in this tissue accompanying Varying amounts of a-lipoic acid were added to the diets at the proliferation of the endoplasmic reticulum(15). In relation the expense of sucrose. This resulted in a decrease in the to this, some studies(16 – 18) have shown that a-lipoic acid energy content of the experimental diets. However, this increases the mRNA expression or activity or the protein con- decrease was very small (less than 0·23 % of the total energy centrations of drug-metabolising enzymes in cultured cells. of the diets). Upon completion of the experimental period, However, information on the effect of a-lipoic acid on drug- the rats were anaesthetised using diethyl ether and killed by metabolising enzymes in experimental animals is lacking. In bleeding from the abdominal aorta, after which the livers

this context, we investigated the effect of dietary a-lipoic were excised. The present study was approved by the . IP address: acid on the gene expression of hepatic enzymes involved in review board of animal ethics of our institute, and we followed drug metabolism in rats in the present study, employing the institute’s guidelines in the care and use of laboratory ani-

DNA microarray analysis, a powerful technique for analysing mals. We read and followed the ARRIVE (Animal Research: 170.106.202.226 the mRNA expression of thousands of genes in a short Reporting In Vivo Experiments) guidelines for animal research period of time. Drug metabolism usually involves many isoen- (http://www.nc3rs.org.uk/page.asp?id ¼ 1357) in conducting zymes differing in substrate speciﬁcity. A considerable number the animal experiment and in preparing the manuscript.

of isoenzymes are relatively irrelevant and only a small , on (19) number are sensitive to dietary factors . Therefore, micro- 28 Sep 2021 at 21:50:59 Affymetrix GeneChip and GeneSpring analyses array-based proﬁling of gene expression helps to clarify the overall picture of the physiological effect of a-lipoic acid on Hepatic RNA was extracted according to the method of the mRNA expression of enzymes involved in drug meta- Chomczynski & Sacchi(22). RNA from the livers of six rats fed bolism and to identify isoenzyme(s) speciﬁcally affected by diets containing 0 and 0·5 % (w/w) a-lipoic acid each was sub- this compound. jected to microarray analyses. Rats with the lowest body

weights in each group at the time of killing were excluded , subject to the Cambridge Core terms of use, available at from the microarray analyses. RNA was processed using kits Materials and methods supplied by Affymetrix to prepare fragmented biotinylated complementary RNA for hybridisation to the Rat Genome Animals and diets 230 2.0 Array. DNA microarray data were analysed using the Male Sprague–Dawley rats obtained from Charles River Japan GeneSpring GX version 7.3 software (Agilent Technologies, at 4 weeks of age were housed individually in animal cages in Inc.). Normalisations per chip and per gene were carried out a room with controlled temperature (20–228C), humidity (55– using the 50th percentile value of signal intensity in each 65 %) and lighting (lights on from 07.00 to 19.00 hours) and chip and the mean values in rats fed a control diet free of fed a commercial non-purified diet (Type NMF; Oriental a-lipoic acid, respectively. After normalisation, the following Yeast Company) for 7 d. After this acclimatisation period, the filtering criteria were adopted to prepare the gene list for sub- rats were randomly divided into four groups with equal sequent analysis to eliminate unreliable data with low signals: mean body weights, consisting of seven animals each, and the expression data had to call more than six P- or M-flags British Journal of Nutrition fed purified experimental diets containing 0, 0·1, 0·25 and according to the Affymetrix algorithm out of twelve measure-

0·5 % (w/w) of R/S-a-lipoic acid for 21 d. Rats are widely ments and the average signal intensity had to be greater than https://www.cambridge.org/core/terms used as an animal model for humans to evaluate the physio- 100 in each treatment. The expression data of 12 335 genes ful- logical activity of dietary factors. The basal composition of filled these criteria. Fold changes in the expression of genes in the experimental diets was as follows (in wt%): palm oil, 10; rats fed the diet containing 0·5 % (w/w) a-lipoic acid casein, 20; maize starch, 15; cellulose, 2; mineral mixture compared with the values in rats fed the diet free of this (AIN-93G-MX)(20), 3·5; vitamin mixture (AIN-93-VX)(20), 1·0; compound were evaluated using this gene list. L-cystine, 0·3; choline bitartrate, 0·25; sucrose, 100 g. There were 15 g of DL-a-tocopherol acetate in 1 kg of AIN-93-VX. Real-time PCR quantification of mRNA HPLC analysis revealed that 1 kg of palm oil contained 0·164 g of D-a-tocopherol and 0·0059 g of D-g-tocopherol. The real-time PCR quantification of mRNA was carried out as

(23) . Therefore, all the experimental diets contained 0·015 % (w/w) described previously in rats fed diets containing 0, 0·1, https://doi.org/10.1017/S0007114514000841 DL-a-tocopherol acetate, 0·016 % (w/w) D-a-tocopherol and 0·25 and 0·5 % (w/w) a-lipoic acid. The abundance of 0·0006 % (w/w) D-g-tocopherol as antioxidants. Although the mRNA was calculated as a ratio to the abundance of b-actin AIN-93G diet formula recommended by the American mRNA in each complementary DNA sample and expressed Institute of Nutrition(20) is suggested to provide 0·0014 % (w/w) as a fold change, assigning a value of 1 for rats fed the diet tert-butylhydroquinone as an antioxidant, we did not free of a-lipoic acid. The mRNA levels of the aldehyde dehy- use this compound in the present study. Chemically drogenase 1 family, member A1 (Aldh1a1), aldo-keto synthesised R/S-a-lipoic acid with purity exceeding 990 g/kg reductase family 1, member D1 (Akr1d1), aldo-keto reductase (manufacturer’s statement) was purchased from Tokyo family 7, member A3 (Akr7a3), cytochrome P450, family 2, Chemical Industry. Enantioselective HPLC analysis(21) using subfamily b, polypeptide 12 (Cyp2b12), cytochrome P450, 11-mercaptoundecanoic acid as an internal standard revealed family 4, subfamily b, polypeptide 1 (Cyp4b1), epoxide hydro- that the R/S-a-lipoic acid preparation contained 45·9 and lase 1, microsomal (Ephx1), ﬂavin-containing monooxygenase Downloaded from

a-Lipoic acid and hepatic drug metabolism 297

5(Fmo5), glutamate–cysteine ligase, modiﬁer subunit (Gclm), distribution of the observations were evaluated using Levene’s https://www.cambridge.org/core glutamate–cysteine ligase, catalytic subunit (Gclc), glutathione test and the Kolmogorov–Smirnov test, respectively. If var- reductase (Gsr), glutathione S-transferase alpha 3 (Gsta3), glu- iances were heterogeneous and/or the distributions were not tathione S-transferase pi 1 (Gstp1), glutathione S-transferase normal, they were transformed logarithmically. The transform- theta 1 (Gstt1), glutathione S-transferase theta 3 (Gstt3), ations were successful in rendering the variance of the obser- metallothionein 1a (Mt1a) and NAD(P)H dehydrogenase, vation constant and the distribution of data normal, and hence quinone 1 (Nqo1) were measured using the TaqMan probe the transformed values were used for subsequent statistical PCR method as described in the handbook (Real-time PCR evaluations. Signiﬁcant differences in the means

Systems Chemistry Guide) supplied by Applied Biosystems. for microarray data at a level of P,0·05 were evaluated . IP address: The nucleotide sequences of primers and probes used for with two-sided Student’s t test. The other data were analysed detecting these mRNA were designed using the Primer Express with one-way ANOVA to establish whether the effect of

Software (Applied Biosystems), and these are listed in Table 1. a-lipoic acid was signiﬁcant, and signiﬁcant differences in 170.106.202.226 The mRNA levels of the ATP-binding cassette, subfamily B, the means at a level of P,0·05 were evaluated with two- member 1A (Abcb1a), ATP-binding cassette, subfamily C, sided Tukey’s test. member 4 (Abcc4), carboxylesterase 2C (Ces2c), glutathione

peroxidase 2 (Gpx2), nuclear receptor subfamily 1, group I, , on

member 3 (Nr1i3), sulfotransferase family, cytosolic, 1C, Results 28 Sep 2021 at 21:50:59 member 2 (Sult1c2) and UDP-glucuronosyltransferase 2 Growth parameters and liver weight family, polypeptide B1 (Ugt2b1) were measured using the SYBR Green PCR method. Forward and reverse primers a-Lipoic acid at a dietary level of up to 0·25 % (w/w) did not used for detecting these mRNA were designed, prepared and affect food intake (Table 2). However, the diet containing provided by Takara Bio, Inc. The reaction speciﬁcity for this 0·5 % (w/w) a-lipoic acid compared with the diet free of this

method was verified by a melting curve analysis. The nucleo- compound significantly reduced this parameter. Growth was , subject to the Cambridge Core terms of use, available at tide sequences of primers are also listed in Table 1. significantly lower in rats fed diets containing 0·25 and 0·5 % (w/w) a-lipoic acid than in those fed the a-lipoic acid-free diet. The diet containing 0·1 % (w/w) a-lipoic acid did not Enzyme assays affect this parameter. Liver weights were the same among About 2·5 g of each liver sample were homogenised with 15 ml rats fed diets containing 0, 0·1 and 0·25 % (w/w) a-lipoic of 0·25 M-sucrose containing 1 mM-EDTA and 3 mM-Tris–HCl acid. However, this parameter was significantly higher in rats (pH 7·2) and centrifuged at 200 000 g for 30 min. The activities fed the diet containing 0·5 % (w/w) a-lipoic acid than in of Gst and Gsr were measured spectrophotometrically using those fed the a-lipoic acid-free diet. 200 000 g supernatant as an enzyme source. The activity of Gst was assayed using 1-chloro-2,4-dinitrobenzene, 1,2- Microarray analysis of mRNA expression dichloro-4-nitrobenzene, p-nitrobenzyl chloride and ethacrynic acid as substrates. The respective conditions (concen- Considerable numbers of genes were affected by dietary a- British Journal of Nutrition trations of substrates and pH) under which enzyme activity lipoic acid. In total, 997 and 1074 of the 12 335 filtered

was determined using these substrates were the same as genes were found to be up-regulated and down-regulated, https://www.cambridge.org/core/terms those described by Habig & Jakoby(24). The activity of Gsr respectively, more than 1·5-fold by the diet containing 0·5 % was measured according to the method described by Carlberg (w/w) a-lipoic acid. The numbers of genes up-regulated and & Mannervik(25). down-regulated more than 2-fold by a-lipoic acid were 410 and 245, respectively. We had previously reported that a-lipoic acid reduces the activity and mRNA levels of many Analyses of serum and liver components enzymes involved in lipogenesis in the liver(12 – 14). Consistent For the determination of hepatic glutathione concentration, with this observation, in the present study using DNA micro- about 1·5 g of liver sample were homogenised with 4·5 ml of array analysis, we observed that a-lipoic acid down-regulated 8 % perchloric acid and centrifuged at 15 000 g for 30 min. many genes related to lipogenesis more than 1·5-fold. These

The supernatants (4 ml each) were neutralised with 3 M- included the patatin-like phospholipase domain containing 3 https://doi.org/10.1017/S0007114514000841

K2CO3. After allowing to stand in ice for 10 min, the mixtures (Pnpla3; four probes detected the mRNA levels of this protein were centrifuged at 3000 g for 10 min. The supernatants were and the values assigning a value of 1 for rats fed the diet free used to determine glutathione concentration by the method of of a-lipoic acid were in the range of 0·052–0·081), fatty acid Anderson(26). Malondialdehyde in the serum and liver samples synthase (Fasn; the values for two probes were 0·125 and was analysed by HPLC as thiobarbituric acid adducts(27). 0·251), acetyl-CoA carboxylase-a and -b (Acaca and Acacb; the values for two probes were 0·350 and 0·438 and 0·166 and 0·188, respectively), ELOVL fatty acid elongase-2, -5 and Statistical analysis -6 (Elovl2,-5 and -6; 0·337 and 0·505 for Elovl2 and -5, respect- Microsoft Excel add-in software (Excel Statistics 2010, Social ively, and 0·213 and 0·250 for Elovl6 for two probes, respect- Survey Research Information Company) was used for statistical ively), pyruvate kinase, liver and red blood cell (Pklr; 0·351), analysis. The constancy of the variance and normality of the thyroid hormone-responsive protein (Thrsp; 0·383 and 0·506 British Journal of Nutrition 298

Table 1. Primers and probes used in real-time PCR analysis of mRNA

Length of PCR GenBank products accession Genes (gene symbol) Sense primer Antisense primer Probe (bp) no.

TaqMan probe PCR method Aldehyde dehydrogenase 1 50-GGAGGACCAGTCGTGATTTAAGC-30 50-CCGACTCTGCAGTTATCATGCA-30 50-TGTCCCTCTGTGACCCCTTGAACTGCT-30 82 NM_022407 family, member A1 (Aldh1a1) Aldo-keto reductase family 1, 50-CACCACATACCCCTAAATGATGGT-30 50-CCGGTCTAGGGTCTGAGTAGGTT-30 50-ACAGCATTCCGATCATCGGGCTTG-30 73 NM_138884 member D1 (Akr1d1) Aldo-keto reductase family 7, 50-GGTTCTTTCCTTTTTAGACAGGTCACT-30 50-CTCAGCCAGCTCTCACTTTGAA-30 50-TTTCTTCCCTGCTTTCTATACAGCCAGTTGC-30 86 NM_013215 member A3 (Akr7a3) Cytochrome P450, family 2, 50-GAACAAAATTATGCTGTGAGTCTAGAAGA-30 50-CAGCTTCGTTATATGAATTTCATCTGA-30 50-CAACCCCCTCTCCTGCTTCTGTTTCA-30 85 NM_017156 subfamily b, polypeptide 12 (Cyp2b12) Cytochrome P450, family 4, 50-GGCCAGGGCTATGGACAGTT-30 50-ATCTCAAGGGCATGACCAAAA-30 50-CCCAGGGCCCCCCACACA-30 66 NM_016999 subfamily b, polypeptide 1 (Cyp4b1) Epoxide hydrolase 1, 50-GAGTTTTACAAGATCATCCCACTACTGA-30 50-TTCAAACACGTGCTCGTCACT-30 50-TGACCCCAAGTCCCACGGTCT-30 72 NM_001034090 microsomal (Ephx1) Flavin-containing mono- 50-TGGGAGCCATTATGCCTATTTC-30 50-GGCAATTTCTTTAGCCCTTTGA-30 50-AGCTCCAAGGACGCTGGGCCACT-30 76 NM_144739 oxygenase 5 (Fmo5) Glu–Cys ligase, modiﬁer 50-CGCCTGCGGAAAAAGTGT-30 50-TCATTCAAGGTCTTTTGGATACAGTCT-30 50-CGTCCACGCACAGCGAGGAGCT-30 71 NM_017305 subunit (Gclm) Glu–Cys ligase, catalytic 50-AACATCAGGCTCTTTGCACGAT-30 50-TGCTCTGGCAGTGTGAATCC-30 50-CTTCATTTCCCAGGCTAGGCTGCCC-30 70 NM_012815 subunit (Gclc) Ide T. Glutathione reductase (Gsr)50-AACATCCCTACCGTGGTCTTCA-30 50-GGACGGCTTCATCTTCAGTGA-30 50-CACCCGCCTATCGGGACAGTGG-30 70 NM_053906 Glutathione S-transferase 50-TAAAGGCCCTGAGAACCAGAGT-30 50-TGGCTGCCAGGCTGAAG-30 50-AGCAACCTCCCCACAGTGAAGAAATT-30 67 NM_001009920 alpha 3 (Gsta3) Glutathione S-transferase pi 1 50-GGAGGAGGTGGTTACCATAGATGT-30 50-GGAGCTGCCCATACAGACAAG-30 50-TGGCTTCAAGGCTCGCTCAAGTCC-30 71 NM_012577 (Gstp1) Glutathione S-transferase 50-CCGTGCTCGTGTGGATGAG-30 50-CAGGGTCCGGAGACAGCTT-30 50-TGGCATGGCAGCATACGACCCTTC-30 70 NM_053293. theta 1 (Gstt1) Glutathione S-transferase 50-TGCAAGGGCAGTCACGAA-30 50-GCCTTTGTCCTCAGGGTCTTC-30 50-AGCCCTGCACCCCACCTATACCA-30 70 BM390378 theta 3 (Gstt3) Metallothionein 1a (Mt1a)50-TGTGCCTGAAGTGACGAACAG-30 50-AGGTGTACGGCAAGACTCTGAGT-30 50-TGCCCTCAGGTGTAAATAATTTCCGGACC-30 80 NM_138826 NAD(P)H dehydrogenase, 50-GCCGAAGGACTCGGAGAAC-30 50-CTCAGGCGGCCTTCCTTATA-30 50-TTCAGTACCCTGTTGAGTCATCTCTG-30 69 NM_017000 quinone 1 (Nqo1) Thioredoxin reductase 1 50-TTTGACAAGAAGGTGATGGTCTTG-30 50-CCCCCGAGACCCCATCT-30 50-ACTTCGTCACACCAACTCCTCTCGGAAC-30 71 NM_031614 (Txnrd1) SYBR Green PCR method ATP-binding cassette, sub- 50-AGATCGTGAAGGCAGCCAAG-30 50-AGAAGTAAGATGTGAGGCTGTCTGA-30 166 NM_133401·1 family B, member 1A (Abcb1a) ATP-binding cassette, sub- 50-AGCCCTGCGGTTATGAGCAC-30 50-GGACACCCTGGAAACAGACTTGA-30 106 NM_133411·1 family C, member 4 (Abcc4) Carboxylesterase 2C (Ces2c)50-AGCTGCTGAGTAGGCGGATGA-30 50-CCAACACAGGCCAGTAGGGTAGA-30 93 NM_133586·1 Glutathione peroxidase 2 50-TGAGTTGCAGTGCCGCTTTC-30 50-GGACATACTTGAGGCTGTTCAGGA-30 116 NM_183403·2 (Gpx2) Nuclear receptor subfamily 1, 50-ATGGAGCAACAGTCAAGACTCCAA-30 50-CCCTGGATGCGATGGATTTC-30 116 NM_022941·3 group I, member 3 (Nr1i3) Sulfotransferase family, 50-ACATGAAGGTGGCCTTTGGAA-30 50-AACTGGCTCCACACTGACCATC-30 81 NM_133547·4 cytosolic, 1C, member 2 (Sult1c2) UDP-glucuronosyltransferase 50-GCTTCTGCTCTTGCCCAAATTC-30 50-GCCTCATAGATGCCATTTGTTCC-30 176 NM_173295·1 2 family, polypeptide B1

(Ugt2b1)

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a-Lipoic acid and hepatic drug metabolism 299

Table 2. Growth parameters and liver weight https://www.cambridge.org/core (Mean values with their standard errors, n7)

Dietary a-lipoic acid (wt%)

0 0·1 0·25 0·5

Mean SEM Mean SEM Mean SEM Mean SEM

Body weight

Initial (g) 136 3 135 2 135 2 138 2 . IP address: Final (g) 309 9 297 10 274* 7 260** 7 Growth (g/21 d) 173 8 162 10 139* 9 122** 7 Food intake (g/d) 19·5 0·8 18·7 0·8 17·8 0·2 15·9** 0·4

Liver weight (g/100 g body weight) 5·15 0·27 4·98 0·16 5·06 0·18 5·86* 0·12 170.106.202.226

Mean values were signiﬁcantly different from those of rats fed the a-lipoic acid-free diet: * P,0·05; ** P,0·01.

for two probes), phosphogluconate dehydrogenase (Pgd; Mt4, have so far been discovered. a-Lipoic acid at a dietary 0·403), stearoyl-CoA desaturase 1 (Scd1; 0·458), glycerol- level of 0·5 % (w/w) caused about a 10-fold increase in the , on 3-phosphate acyltransferase, mitochondrial (Gpam; 0·467 mRNA expression of Mt1a and Mt2A. In organisms, two sys- 28 Sep 2021 at 21:50:59 and 0·619 for two probes), glucose-6-phosphate dehydrogen- tems are considered critical for general reduction–oxidation ase (G6pd; 0·504), ATP citrate lyase (Acly; 0·519 and 0·532 for (redox) regulation, the thioredoxin and glutaredoxin (Glrx)/ two probes), fatty acid desaturase-1 and -2 (Fads1 and -2; glutathione systems(30). Thioredoxin and thioredoxin 0·566 and 0·541, respectively), malic enzyme 1 (Me1; 0·561 reductase and Glrx, glutathione and Gsr represent primary and 0·600 for two probes) and SREBF chaperone (Scap; 0·575). components of the former and latter systems, respectively. , subject to the Cambridge Core terms of use, available at In addition to the lipogenic pathway, we found that genes In rats, three forms of thioredoxin reductase have been ident- involved in drug metabolism and the antioxidation system ified (Txnrd1–3). a-Lipoic acid caused more than a 2-fold were targeted by dietary a-lipoic acid. Table 3 lists the increase in the mRNA expression of Txnrd1 in the present genes affected more than 1·5-fold by the diet containing study. Among the proteins involved in the Glrx/glutathione 0·5 % (w/w) a-lipoic acid. The drug metabolism pathway system, a-lipoic acid increased the mRNA expression of Gsr, has generally been grouped into phase I reactions, in which Gclc (a heavy catalytic subunit of glutamate–cysteine ligase enzymes carry out oxidation, reduction or hydrolysis reac- that catalyses the first step of glutathione synthesis) and the tions; phase II reactions, in which enzymes form a conjugate mitochondrial isoform of Glrx (Glrx2), but unexpectedly of the substrate; and phase III reactions, in which enzymes decreased the mRNA expression of the cytosolic form of transport drugs. a-Lipoic acid apparently increased the Glrx (Glrx1). Although the magnitude of the increase did mRNA expression of many phase I enzymes that catalyse oxi- not exceed 1·5-fold, a-lipoic acid also caused a significant dation (Cyp4b1, Cyp2b12 and Fmo5), reduction (Akr7a3,

British Journal of Nutrition increase in the mRNA expression of a light regulatory subunit Aldh18a1, Aldh1a7, Akr1c1, Aldh1a1, Akr1d1 and Nqo1) of glutamate–cysteine ligase (Gclm) (1·00 (SEM 0·056) and 1·45 and hydrolysis reactions (Ces2c and Ephx1). This compound (SEM 0·09) in rats fed the a-lipoic acid-free diet and the diet https://www.cambridge.org/core/terms also increased mRNA expressions of phase II enzymes that containing 0·5 % (w/w) a-lipoic acid, respectively). The perox- catalyse glutathione conjugation (Gsta3, Gstt3, Gstp1, Gstt1 iredoxins function as cysteine-dependent thiol peroxidases and Gsto1), sulfation (Sult1c2) and glucuronidation (Ugt2b1 that detoxify H O and lipid peroxides and hence exert and Ugt1a). The mRNA expression of some phase III transpor- 2 2 cytoprotective and antioxidative effects. Under increased oxi- ters, including Abcb1a, ATP-binding cassette, subfamily B, member 1B (Abcb1b), Abcc4 and ATP-binding cassette, sub- dative stress, the cysteine moiety of peroxiredoxins is hyper- family A (ABC1), member 8 (Abca8), was also up-regulated oxidised to sulﬁnic acid and hence they lose their by a-lipoic acid. Nr1i3 is a member of the nuclear receptor peroxidase activity. This inactive form can be reduced back superfamily and activates many genes involved in drug meta- to the active thiol form by sulﬁredoxin 1 (Srxn1), an ATP- (31) (28) dependent reductase . The mRNA expression of this .

bolism . a-Lipoic acid increased the mRNA expression of https://doi.org/10.1017/S0007114514000841 this transcription factor. reductase was found to be up-regulated by a-lipoic acid in We also observed that a-lipoic acid increased the mRNA the present study. APEX nuclease 1 (Apex1) is a DNA repair expression of miscellaneous proteins involved in the antioxi- protein that also functions as a redox regulator in mam- (30) dation system. Metallothionein is a cysteine-rich protein that mals . a-Lipoic acid caused a 2·5-fold increase in the has marked capacity to bind metal ions. This protein controls mRNA expression of this protein. Glutathione peroxidase metal ion homeostasis and prevents the toxicity of heavy catalyses the reduction of hydroperoxides and H2O2 by gluta- metals. Metallothionein has also been found to perform thione and thereby protects against oxidative damage. So far, diverse functions. In particular, this protein has been eight different isoforms of glutathione peroxidase have considered to be a key component of cellular defence against been identiﬁed in humans (GPX1–8) and rodents (Gpx1–8). oxidative stress(29). In rats, four types of the metallothionein Microarray analysis indicated that a-lipoic acid increased the gene, termed Mt1a, metallothionein 2A (Mt2A), Mt3 and mRNA expression of Gpx2 but not of other Gpx isoforms. British Journal of Nutrition

Table 3. Microarray analysis results for the mRNA of hepatic proteins involved in drug metabolism and prevention of oxidative stress 300 (Mean values with their standard errors, n6)

Fold changes (dietary a-lipoic acid, wt%)

00·5

Accession no. Gene symbol Gene name Mean SEM Mean SEM

Oxidation, reduction and hydrolysis M29853 Cyp4b1 Cytochrome P450, family 4, subfamily b, polypeptide 1 1·00 0·07 14·9** 1·7 NM_013215 Akr7a3 Aldo-keto reductase family 7, member A3 (aﬂatoxin aldehyde reductase) 1·00 0·08 8·51** 0·33 AI454613 Cyp2b12 Cytochrome P450, family 2, subfamily b, polypeptide 12 1·00 0·06 3·80** 0·19 BG380693 Aldh18a1 Aldehyde dehydrogenase 18 family, member A1 1·00 0·08 4·12** 0·46 M23995 Aldh1a7 Aldehyde dehydrogenase family 1, subfamily A7 1·00 0·31 3·52** 0·50 NM_022407 Aldh1a1 Aldehyde dehydrogenase 1 family, member A1 1·00 0·19 2·77** 0·20 AI454611 Fmo5 Flavin-containing monooxygenase 5 1·00 0·19 2·60** 0·36 AI028867 Akr1d1 Aldo-keto reductase family 1, member D1 1·00 0·13 1·99** 0·09 J02679 Nqo1 NAD(P)H dehydrogenase, quinone 1 1·00 0·15 1·92** 0·14 NM_133586 Ces2c Carboxylesterase 2C 1·00 0·16 1·93** 0·17 AI072107 Akr1c1 Aldo-keto reductase family 1, member C1 1·00 0·22 1·76** 0·36 NM_012844 Ephx1 Epoxide hydrolase 1 1·00 0·04 1·76** 0·09 Conjugation AA945082 Gsta3 Glutathione S-transferase alpha 3 1·00 0·26 51·7** 3·4 BI300997 Sult1c2 Sulfotransferase family, cytosolic, 1C, member 2 1·00 0·15 13·5** 1·5 NM_133547 Sult1c2 Sulfotransferase family, cytosolic, 1C, member 2 1·00 0·18 8·86** 0·8 BM390378 Gstt3 Glutathione S-transferase, theta 3 1·00 0·20 4·83** 0·86

X02904 Gstp1 Glutathione S-transferase pi 1 1·00 0·05 3·45** 0·86 Ide T. M13506 Ugt2b17 UDP-glucuronosyltransferase 2 family, polypeptide B17 1·00 0·17 1·94* 0·46 NM_053293 Gstt1 Glutathione S-transferase theta 1 1·00 0·09 1·87** 0·24 J02612 Ugt1a1; Ugt1a6; Ugt1a7; UDP-glucuronosyltransferase 1 family, polypeptide A1; A6; A7; A8; 1·00 0·05 1·66** 0·06 Ugt1a8; Ugt1a3; Ugt1a10 A2; A3; A10 BE113459 Gsto1 Glutathione S-transferase omega 1 1·00 0·04 1·62** 0·07 Transporters AF286167 Abcb1a ATP-binding cassette, subfamily B (MDR/TAP), member 1A 1·00 0·15 6·62** 0·67 AF257746 Abcb1a ATP-binding cassette, subfamily B (MDR/TAP), member 1A 1·00 0·17 3·53** 0·72 AY082609 Abcb1b; Abcb1a ATP-binding cassette, subfamily B (MDR/TAP), member 1B; 1A 1·00 0·10 3·45** 0·34 BE100533 Abcc4 ATP-binding cassette, subfamily C (CFTR/MRP), member 4 1·00 0·09 3·00** 0·23 BF386852 Abca8 ATP-binding cassette, subfamily A (ABC1), member 8 1·00 0·19 2·49** 0·26 Transcription factor NM_022941 Nr1i3 Nuclear receptor subfamily 1, group I, member 3 1·00 0·18 2·59** 0·13 Antioxidation system AF411318 Mt1a Metallothionein 1a 1·00 0·22 11·6** 0·6 BM383531 Mt2A Metallothionein 2A 1·00 0·13 9·00** 1·56 H31896 Srxn1 Sulﬁredoxin 1 1·00 0·05 5·51** 0·86 AA800587 Gpx2 Glutathione peroxidase 2 1·00 0·18 4·20** 0·35 AF311054 Apex1 APEX nuclease (multifunctional DNA repair enzyme) 1 1·00 0·06 2·54** 0·22 U63923 Txnrd1 Thioredoxin reductase 1 1·00 0·05 2·43** 0·17 NM_031614 Txnrd1 Thioredoxin reductase 1 1·00 0·04 2·20** 0·14 NM_053906 Gsr Glutathione reductase 1·00 0·07 1·94** 0·10 AW525635 Glrx2 Glutaredoxin 2 1·00 0·04 1·67** 0·12 J05181 Gclc Glu–Cys ligase, catalytic subunit 1·00 0·04 1·65** 0·12 AA848536 Glrx2 Glutaredoxin 2 1·00 0·09 1·56** 0·20 NM_012580 Hmox1 Haeme oxygenase (decycling) 1 1·00 0·10 1·52** 0·10 NM_022278 Glrx1 Glutaredoxin 1 1·00 0·07 0·438** 0·032

Mean values were signiﬁcantly different from those of rats fed the a-lipoic acid-free diet: * P,0·05; ** P,0·01.

a-Lipoic acid and hepatic drug metabolism 301

Real-time PCR analysis of mRNA expression and induced by a-lipoic acid was considerably lower when the https://www.cambridge.org/core determination of enzyme activities values were obtained using real-time PCR than when obtained using DNA microarray analysis. The observations made with DNA microarray analysis As a-lipoic acid increased the mRNA expression of some Gst revealed that dietary a-lipoic acid increased the mRNA isoenzymes, we analysed the activity of Gst using four different expression of proteins involved in drug metabolism and substrates (1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitro- attenuation of oxidative stress. To conﬁrm this observation, we analysed mRNA expression by real-time PCR analysis in benzene, p-nitrobenzyl chloride and ethacrynic acid). The enzyme activity measured using 1-chloro-2,4-dinitrobenzene

rats fed diets containing varying amounts of a-lipoic acid. . IP address: The observations made with real-time PCR analysis essentially as a substrate was signiﬁcantly higher in rats fed diets contain- conﬁrmed those made with DNA microarray analysis. In fact, ing various amounts of a-lipoic acid than in those fed the diet a-lipoic acid dose dependently increased the mRNA free of this compound (Fig. 3). The activity of the enzyme

170.106.202.226 expression of phase I enzymes (Cyp2b12, Cyp4b1, Fmo5, measured using this substrate was about 1·5 times higher in Akr1d1, Akr7a3, Aldh1a1, Nqo1, Ces2c and Ephx1) (Fig. 1), rats fed the diet containing 0·5 % (w/w) a-lipoic acid than in phase II enzymes (Gsta3, Gstp1, Gstt1, Gstt3, Ugt2b1 and those fed the diet free of this compound. Signiﬁcant but less Sult1c2) and phase III transporters (Abcb1a and Abcc4) prominent increases in enzyme activity were also observed

, on (Fig. 2). We also conﬁrmed that a-lipoic acid even at a dietary when 1,2-dichloro-4-nitrobenzene was used as a substrate in

28 Sep 2021 at 21:50:59 level of 0·1 % (w/w) signiﬁcantly increased the mRNA rats fed diets containing 0·25 and 0·5 % (w/w) a-lipoic acid expression of Nrli3. The magnitudes of the increase observed than in those fed the a-lipoic acid-free diet. However, signiﬁ- in rats fed the diet containing 0·5 % (w/w) a-lipoic acid gene- cant increases in enzyme activity in rats fed diets containing rally approximated those observed with DNA microarray various amounts of a-lipoic were not observed when using analysis except for a few genes (Ces2c, Gsta3 and Sult1c2). p-nitrobenzyl chloride and ethacrynic acid as substrates

The extent of changes in the expression of these genes (data not shown). , subject to the Cambridge Core terms of use, available at

(a)Cyp2b12 (b)Cyp4b1 (c)Fmo5 (d)Akr1d1 (e) Akr7a3 ** * ** 2·0 10 ** ** ** 20 3 * 3 8 ** 1·5 15 2 6 2 1·0 10 ** ** 4

British Journal of Nutrition 1 1 0·5 ** 5 2

https://www.cambridge.org/core/terms * 0 0 0 0·0 0

(f)Aldh1a1 (g)Aldh1a7 (h)Nqo1 (i)Ces2c (j) Ephx1 ** ** 3 ** 2·5 2 ** ** 2·0 ** 1·5 ** **

mRNA level (fold change) ** 2·0 * 1·5 ** 2 ** 1·5 1·0 1·5

https://doi.org/10.1017/S0007114514000841 1·0 1 1·0 1 0·5 0·5 0·5 0·5

0 0·0 0·0 0·0 0·0 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 α-Lipoic acid (wt%) Fig. 1. Levels of the mRNA of enzymes involved in phase I reactions in drug metabolism in the liver of rats fed diets containing various amounts of a-lipoic acid. The levels of mRNA were quantiﬁed using real-time PCR analysis. (a) Cyp2b12,(b)Cyp4b1, (c) Fmo5,(d)Akr1d1,(e)Akr7a3, (f) Aldh1a1,(g)Aldh1a7,(h)Nqo1, (i) Ces2c, (j) Ephx1. Values are means, with their standard errors represented by vertical bars (n 7). Mean values were signiﬁcantly different from those of rats fed the a-lipoic acid-free diet: * P,0·05; ** P,0·01. The names of genes represented by gene symbols are given in Table 3. Downloaded from

302 T. Ide

(a) Gsta3 (b)Gstp1 (c)Gstt1 (d) Gstt3 https://www.cambridge.org/core ** 5 ** ** 2·0 ** 15 5 ** 4 1·5 4 ** 10 3 3 1·0

2 . IP address: ** * 2 5 ** 0·5 1 1

170.106.202.226

0 0 0·0 0 (e)Ugt2b1 (f)Sult1c2 (g)Abcb1a (h) Abcc4 (i) Nrli3 2·5 ** ** ** **

** , on 4 * 3 **

28 Sep 2021 at 21:50:59 2·0 4 ** 2·0 mRNA level (fold change) 3 ** ** 1·5 3 2 1·5

2 * 1·0 2 ** 1·0

, subject to the Cambridge Core terms of use, available at 1 1 0·5 1 0·5

0·0 0 0 0 0·0 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 α-Lipoic acid (wt%) Fig. 2. Levels of the mRNA of enzymes involved in phase II conjugation reactions and phase III transportation in drug metabolism as well as a transcription factor regulating the gene expression of drug-metabolising enzymes in the liver of rats fed diets containing various amounts of a-lipoic acid. The levels of mRNA were quantiﬁed using real-time PCR analysis. (a) Gsta3,(b)Gstp1, (c) Gstt1,(d)Gstt3,(e)Ugt2b1, (f) Sult1c2,(g)Abcb1a,(h)Abcc4 and (i) Nr1i3. Values are means, with their standard errors represented by vertical bars (n 7). Mean values were signiﬁcantly different from those of rats fed the a-lipoic acid-free diet: * P,0·05; ** P,0·01. The names of genes represented by gene symbols are given in Table 3.

In Fig. 4(a), the effect of dietary a-lipoic acid on the mRNA of this enzyme. Consistent with the observation that British Journal of Nutrition levels of proteins involved in the thiol redox regulation system a-lipoic acid increased the mRNA expression of proteins

and Mt1a analysed by real-time PCR analysis among rats fed involved in the redox system, a-lipoic acid strongly and https://www.cambridge.org/core/terms diets containing varying amounts of a-lipoic acid is shown. dose dependently reduced the concentrations of malondial- The results obtained using DNA microarray analysis were gen- dehyde, a marker of lipid peroxidation, in both the serum erally conﬁrmed using real-time PCR analysis; that is, dietary and liver (Fig. 4(d)). a-lipoic acid dose dependently increased the mRNA levels of Gsr, Gpx2, Gclc, Gclm, Glrx2, Txnrd1, Srnx1 and Apex1. Discussion The decrease in the mRNA expression of Glrx1 caused by the diet containing 0·5 % (w/w) a-lipoic acid observed using Previous studies have indicated that a-lipoic acid increases the DNA microarray analysis was also observed using real-time mRNA expression or the activity or protein levels of enzymes PCR analysis. However, the decrease was considerably attenu- involved in drug metabolism, including NAD(P)H:quinone

(16 – 18) . ated when the parameter was analysed using real-time PCR oxidoreductase , Gst-a2 and microsomal epoxide hydro- https://doi.org/10.1017/S0007114514000841 analysis. A significant decrease in this parameter was not lase(18), in various types of cultured cells. However, an animal confirmed in rats fed diets containing 0·1 and 0·25 % (w/w) study examining the physiological activity of a-lipoic acid a-lipoic acid. As a-lipoic acid increased the mRNA expression affecting drug-metabolising enzymes has been lacking. of subunits of a rate-limiting enzyme in glutathione The present study using DNA microarray analysis unequivo- biosynthesis, we measured hepatic glutathione concentration cally demonstrated that dietary a-lipoic acid up-regulates the (Fig. 4(b)). Dietary a-lipoic acid dose dependently increased gene expression of various proteins involved in drug meta- this parameter, and values observed in rats fed diets bolism. Drug metabolism involves many enzymes that catalyse containing 0·25 and 0·5 % (w/w) a-lipoic acid were numerous types of chemical reactions, and each reaction is significantly higher than those in rats fed the diet free of often catalysed by many isoenzymes differing in substrate a-lipoic acid. We also measured the activity of Gsr (Fig. 4(c)). specificity. Therefore, the DNA microarray technique is quite Dietary a-lipoic acid dose dependently increased the activity useful for identifying isoenzymes involved in drug metabolism Downloaded from

a-Lipoic acid and hepatic drug metabolism 303

(a) (b) It has been suggested that various Akr isoenzymes are https://www.cambridge.org/core ** ** involved in the detoxiﬁcation of lipid aldehydes and various 40 1500 ** ** xenobiotic compounds(44). DNA microarray and real-time 30 PCR analyses revealed that a-lipoic acid dose dependently 1000 20 increased the mRNA expression of Akr7a3, Akr1d1 and 500 Akr1c1. Akr7a3, alternatively known as aﬂatoxin aldehyde

Enzyme activity 10 reductase, was discovered(45) as an enzyme to reduce aﬂa-

(nmol/min per mg protein) 0 0 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 toxin B1-dihydrodiol to the dialcohol metabolite in rats.

(46,47) . IP address: α-Lipoic acid (wt%) Studies have demonstrated that this enzyme also reduces various aldehyde and keto substrates. In addition, Fig. 3. Activity of glutathione transferase in the liver of rats fed diets contain- (46 – 48) ing various amounts of a-lipoic acid. Enzyme activity was measured using studies have shown that many compounds having che-

(a) 1-chloro-2,4-dinitrobenzene and (b) 1,2-dichloro-4-nitrobenzene as sub- mopreventive properties increase the expression of this 170.106.202.226 strates. Values are means, with their standard errors represented by vertical enzyme. Studies have indicated that Akr1c1 plays a crucial bars (n 7). Mean values were signiﬁcantly different from those of rats fed the role in the metabolism of xenobiotic compounds and in che- a-lipoic acid-free diet: * P,0·05; ** P,0·01. moprevention(49). Akr1d1 may play a crucial role in bile acid (50) synthesis rather than in drug metabolism. Data suggest , on speciﬁcally affected by various physiological and dietary that several Akr isoforms convert polycyclic aromatic hydro- 28 Sep 2021 at 21:50:59 factors. carbons into carcinogenic compounds, referred to as remote The Cyp proteins are a superfamily of enzymes highly quinones(51). This does not comply with the consideration expressed in the liver and involved in the metabolism of xeno- that Akr are involved in chemoprevention. biotic compounds, as well as the synthesis and catabolism of Carboxylesterases comprise a superfamily of microsomal endogenous compounds. Among the various Cyp, DNA micro- enzymes that catalyse the hydrolysis of a variety of ester-

array and real-time PCR analyses revealed that a-lipoic acid , subject to the Cambridge Core terms of use, available at and amide-containing chemicals to their respective free dose dependently increased the mRNA expression of acids. They are involved in the detoxification or metabolic Cyp4b1 and Cyp2b12. Cyp4b1 is involved in the v-oxidation activation of xenobiotic compounds(52). Dietary a-lipoic acid of lauric acid and mutagenic activation of some chemical increased the mRNA expression of Ces2c but not of other iso- compounds(32,33). Some xenobiotic compounds, such as enzymes. Ces2 isoforms can hydrolyse various xenobiotic terephthalic acid, have been shown to increase the mRNA (52) (34) compounds . Therefore, the increase in the mRNA expression of Cyp4b1 in the liver , and caffeic acid has expression of a member of the Ces2 family may stimulate been shown to increase the mRNA expression in the (35) the metabolism of xenobiotic compounds. colon . The role of Cyp2b12 in hepatic drug metabolism is More than twenty Gst isoenzymes have so far been ident- not clear. ified in rats. In spite of the fact that the DNA microarray con- Fmo are another superfamily of enzymes involved in drug tained nineteen probes for various Gst, we observed that the (36) metabolism and are expressed at high levels in the liver. diet containing 0·5 % (w/w) a-lipoic acid significantly Many different enzyme genes have been identified in mam- increased the mRNA expression of Gsta3, Gstt1 and Gstt3, British Journal of Nutrition mals. Studies have indicated that Fmo1, Fmo3 and Fmo5 are Gstp1 and Gsto1 but not of other Gst more than 1·5-fold.

the major Fmo in mouse liver and Fmo5 predominates over Therefore, the effect of a-lipoic acid on the mRNA expression https://www.cambridge.org/core/terms (37) the other two isoforms . DNA microarray analysis revealed of Gst was again isoenzyme specific. It has been reported that that the mRNA expression of Fmo5 but not of other Fmo several synthetic or natural compounds with anti-carcinogenic (37,38) was up-regulated by dietary a-lipoic acid. Some studies activities increase the mRNA or protein expression of Gsta3 have shown that Fmo5, similar to other Fmo, can oxidise and Gstp1 in rat liver(47,53). So, it is possible that these iso- various endogenous and xenobiotic substrates. forms play a role in the detoxification of xenobiotic com- Many aldehydes generated during normal physiological pounds and cancer prevention. Also, some epidemiological processes are cytotoxic and carcinogenic. Aldh detoxify surveys have indicated that GSTT1 deletion in humans various aldehydes. Aldh is a superfamily consisting of many increases the risk of cancer(54). Gstt3 has so far been identified genes. In the present study, a-lipoic acid increased the in mice and rats and is highly expressed in the liver(55). How-

mRNA expression of Aldh1a1, Aldh1a7 and Aldh18a1. ever, the physiological significance of this Gst isoenzyme in https://doi.org/10.1017/S0007114514000841 Studies have indicated that Aldh1a1 is involved in the detoxi- detoxification and cancer prevention is not known. Gsto1 is fication of various xenobiotic aldehydes(39). As Aldh1a1 and expressed abundantly in the liver, macrophages, and glial Aldh1a7 appear to be coordinately regulated(40,41),itis and endocrine cells(56), but information regarding the role of plausible that not only Aldh1a1 but also Aldh1a7 plays a this isoform in drug metabolism is scarce. A case–control crucial role in drug metabolism. The role of Aldh18a1 in study failed to find a clear-cut relationship between GSTO1 drug metabolism is not clear at present. polymorphisms and cancer risk(57). We observed that dietary The role of Nqo1 and Ephx1 in drug metabolism has been a-lipoic acid increased cytosolic Gst activity when using well documented(42,43). In the present study, we observed 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene that a-lipoic acid increased the mRNA expression of these as substrates. However, the extent of the increases was enzymes. This is agreement with the results of previous much lower than that observed for the mRNA levels of studies in cultured cells(16 – 18). Gsta3, Gstt1 and Gstt3, Gstp1 and Gsto1. This is not surprising Downloaded from

304 T. Ide

(a) (b) (c) (d) (e) https://www.cambridge.org/core Gsr Gpx2 Gclc Gclm Glrx1 ** ** ** 2·0 4 2·0 2·0 ** 1·2 ** ** ** 1·0 1·5 3 1·5 1·5 ** ** ** * 0·8 * 1·0 2 1·0 1·0 0·6 0·4 0·5 1 0·5 0·5 0·2

0·0 0 0·0 0·0 0·0 . IP address:

(f) (g) (h) (i) (j) Glrx2 Txnrd1 Srnx1 Apex1 Mt1a ** ** ** 3 ** ** 1·5 ** 2·0 8 2·0 ** 170.106.202.226 ** ** 1·5 1·5 2 6 1·0 ** ** ** mRNA level (fold change) 1·0 * 1·0 4 0·5 1

0·5 0·5 2 , on

0·0 0·0 0 0·0 0 28 Sep 2021 at 21:50:59 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 α-Lipoic acid (wt%) Glutathione Glutathione reductase Malondialdehyde (k) (l) (m) (n)

** 100 ** Serum 160 Liver 6 ** **

6 , subject to the Cambridge Core terms of use, available at ** mol/g) mol/l) µ 80 µ 120

4 60 4 ** ** ** 80 40 ** ** 2 2

Enzyme activity 40 20 (nmol/min per mg protein) Liver concentration (nmol/g) Serum concentration (

Hepatic concentration ( 0 0 0 0 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 0·0 0·1 0·25 0·5 α-Lipoic acid (wt%) α-Lipoic acid (wt%) α-Lipoic acid (wt%) Fig. 4. Levels of the mRNA of proteins involved in the antioxidation system ((a) Gsr,(b)Gpx2, (c) Gclc,(d)Gclm,(e)Glrx1, (f) Glrx2,(g)Txnrd1,(h)Srnx1, (i) Apex1, (j) Mt1a), (k) concentration of glutathione, (l) activity of glutathione reductase in the liver as well as concentrations of malondialdehyde in the (m) serum and (n) liver of rats fed diets containing various amounts of a-lipoic acid. The levels of mRNA were quantiﬁed using real-time PCR analysis. Values are means,

British Journal ofwith Nutrition their standard errors represented by vertical bars (n 7). Mean values were signiﬁcantly different from those of rats fed the a-lipoic acid-free diet: * P,0·05; ** P,0·01. The names of genes represented by gene symbols are given in Table 3.

https://www.cambridge.org/core/terms because the effect of a-lipoic acid on Gst was isoenzyme glucuronosyltransferase 2 family, polypeptide B17 (Ugt2b17) specific and it did not affect the mRNA levels of many other and isoenzyme(s) belonging to the Ugt1a family. Ugt belong- Gst. a-Lipoic acid failed to affect enzyme activity when using ing to 1a and 2b families are abundantly expressed in the liver p-nitrobenzyl chloride and ethacrynic acid as substrates. The and catalyse the glucuronidation of various endogenous and substrate specificity of each Gst isoenzyme differs consider- xenobiotic compounds(59). ably(56). Therefore, it is reasonable that a-lipoic acid did not The Abc transporters transport a wide variety of exogenous increase the amount of Gst molecules, which is specific to and endogenous substances across membranes. There are p-nitrobenzyl chloride and ethacrynic acid. seven subfamilies, termed Abca, Abcb, Abcc, Abcd, Abce, Cytosolic Sult are phase II drug-metabolising enzymes. In Abcf and Abcg. Many Abc transporters are involved in the

mammals, four to five gene families of Sult have been ident- export of xenobiotic compounds and hence in drug resist- https://doi.org/10.1017/S0007114514000841 ified(58). Each family comprises more than one member, and ance. In the present study, a-lipoic acid increased the mRNA the DNA microarray used in the present study contained expression of three Abc transporters (one each from Abca, seven probes for various Sult1 family members, five for Sult2 Abcb and Abcc subfamilies). As these Abc subfamilies play family members, and one each for Sult4 and Sult5 members. crucial roles in the export of xenobiotics out of the cell(60), Among the various Sult, a-lipoic acid increased the mRNA a-lipoic acid may stimulate drug metabolism through the expression of Sult1c2, but not of the other isoforms. It has up-regulation of not only phase I and II enzymes but also been reported that phenolic compounds including carcino- phase III transporters. gens can serve as substrates for Sult belonging to the 1c a-Lipoic acid increased the mRNA expression of family(58). Nr1i3, alternatively known as constitutive androstane/ Among the various Ugt isoenzymes, a-lipoic acid activated receptor (Car). This transcription factor activates significantly increased the mRNA expression of UDP various genes involved in drug metabolism(28). Downloaded from

a-Lipoic acid and hepatic drug metabolism 305

Therefore, up-regulationof the mRNA expression of this transcrip- system observed in the present study, but also the reduction https://www.cambridge.org/core tion factor may account for the a-lipoic acid-dependent increase in in lipid biosynthesis reported previously(12 – 14). the mRNA levels of various proteins involved in drug metabolism. Finlay et al.(67) have recently reported the results of DNA In the present study, we demonstrated that a-lipoic acid microarray analysis on the hepatic gene expression of young increases the gene expression of proteins involved in the anti- and old rats fed a-lipoic acid. Consistent with our previous oxidation system in addition to that of those involved in drug studies(12 – 14) and the present study, they found that a-lipoic metabolism. The observations that a-lipoic acid increased acid strongly reduced the mRNA levels of proteins involved hepatic glutathione concentration and mRNA levels of two in lipogenesis. They also suggested that a-lipoic acid altered

subunits of g-glutamylcysteine ligase (Gclc and Gclm) were the expression of genes governing circadian rhythm. How- . IP address: in agreement with those made by Suh et al.(61).Xuet al.(8) ever, they did not make any comment regarding the effect have also recently reported that a-lipoic acid increases hepatic of a-lipoic acid on proteins involved in drug metabolism

glutathione concentration and activities of enzymes involved and in the antioxidation system. 170.106.202.226 in the antioxidation system. Moreover, in the present study Previous animal experiments(8 – 11,63) using rats or mice have using DNA microarray analysis, we demonstrated that employed dietary levels of a-lipoic acid of 0·05–1 % (w/w), a-lipoic acid increases the mRNA expression of various which are similar to the levels employed in the present (68) other proteins presumed to be involved in the thiol redox study. A nutritional survey conducted in the UK in 2008–9 , on

system, including Srxn1, Gpx2, Apex1, Txnrd1, Gsr and has indicated the median food intake of adult humans to be 28 Sep 2021 at 21:50:59 Glrx2 (cytosolic form of Glrx), but decreases that of the mito- 435 and 338 g/d for men and women, respectively. A nutri- chondrial form of Glrx (Glrx1). However, real-time PCR tional survey conducted in Japan in 2011 (http://www.mhlw. analysis indicated that a-lipoic acid is rather weak in terms go.jp/stf/houdou/2r9852000002q1st-att/2r9852000002q1wo.pdf) of affecting the mRNA expression of Glrx1. In addition to has also indicated the average food intake of adult humans to the proteins involved in the thiol redox system, a-lipoic be 447 and 374 g/d for men and women, respectively. There-

acid signiﬁcantly increased the mRNA expression of fore, the intake of diets containing 0·1, 0·25 and 0·5 % (w/w) , subject to the Cambridge Core terms of use, available at metallothioneins (Mt1a and Mt2A). Studies have indicated a-lipoic acid used in the present animal study corresponds that metallothionein plays a crucial role in the prevention of to the daily consumption of approximately 340–450, oxidative stress in animals(29). Haeme oxygenase is involved 850–1100 and 1700–2200 mg/person of this compound, in the conversion of haeme into strong antioxidants (biliverdin respectively, when extrapolated to humans. These values are and biliverdin)(62). Therefore, haeme oxygenase is considered comparable to those employed in previous human studies to play a decisive role in the attenuation of the oxidative pro- (200–2400 mg/d per person)(2). With regard to the safe dose

cess. So far, two forms of haeme oxygenase (Hmox1 and level of a-lipoic acid, the acute LD50 (lethal dose 50 %) Hmox2) have been identiﬁed in both humans and rodents. appeared to be very high in rats (.2000 mg/kg body Hmox1 but not Hmox2 is considered to play a crucial role weight), although lower values have been reported in some in protection against oxidative stress(62). other species (dogs, mice and cats) and the NOAEL (no Some previous studies(61,63,64) have indicated that a-lipoic observed adverse effect level) has been estimated to be acid increases the expression of genes involved in the anti- 61·9 mg a-lipoic acid/kg body weight per d for rats(2).On British Journal of Nutrition oxidation system. The present study extended this evidence the basis of the body weight and food intake of rats observed

and newly identified many genes involved in the antioxidation in the present study, approximate dose levels of a-lipoic acid https://www.cambridge.org/core/terms system that are affected by a-lipoic acid. Therefore, it is were estimated to be 16, 40 and 80 mg/kg body weight per d plausible that this at least partly accounts for the potent anti- for animals fed diets containing this compound at the levels of oxidation activity of dietary a-lipoic acid, as confirmed in 0·1, 0·25 and 0·5 % (w/w), respectively. Therefore, these the present study using malondialdehyde as a marker of values did not markedly exceed the NOAEL of a-lipoic acid, lipid peroxidation. although dietary levels of 0·25 and 0·5 % (w/w) significantly A transcription factor called nuclear factor, erythroid derived reduced the body weight of animals. The diet containing 2, like 2 (Nfe2l2) or NFE2-related factor 2 (Nrf2) regulates the 0·25 % (w/w) a-lipoic acid reduced the growth of rats without gene expression of many enzymes involved in the antioxida- significantly affecting food intake in the present study. In this tion system and drug metabolism(61,65). There is a possibility context, it has been reported that a-lipoic acid increases

(69,70) . that this transcription factor is involved in a-lipoic acid-depen- energy expenditure and reduces intestinal sugar absorp- https://doi.org/10.1017/S0007114514000841 dent changes in the mRNA expression of proteins involved in tion(71) in rats. drug metabolism and the antioxidation system. In fact, the It has been reported that orally administered a-lipoic acid in mRNA expression of many genes targeted by this transcription rats is well absorbed from the intestine(2,72). Pharmacokinetic factor, such as Nqo1, Hmox1, subunits of glutamate–cysteine analyses of the metabolic fate of orally and intravenously ligase (Gclm and Gclc), Ephx1, Srxn1 and Abcc4, was up- administered radiolabelled a-lipoic acid have indicated that regulated by dietary a-lipoic acid. Moreover, recent studies 66 % of orally administered a-lipoic acid is absorbed from have indicated that Nrf2 is also involved in the regulation of the intestine. An alternative evaluation based on the measure- hepatic lipid biosynthesis(66). Therefore, alterations in the ments of urinary excretion of 14C gave a higher value of Nrf2 signalling pathway may account for not only the absorption (93 %)(72). a-lipoic acid-dependent changes in the mRNA expression of In conclusion, DNA microarray and real-time PCR analyses proteins involved in drug metabolism and the antioxidation revealed that dietary a-lipoic acid strongly increases the Downloaded from

306 T. Ide

mRNA expression of various proteins involved in drug 10. Song KH, Lee WJ, Koh JM, et al. (2005) a-Lipoic acid https://www.cambridge.org/core metabolism and the antioxidation system. Therefore, it is prevents diabetes mellitus in diabetes-prone obese rats. expected that a-lipoic acid would be effective at ameliorating Biochem Biophys Res Commun 326, 197–202. pathological conditions induced by physiological factors and 11. Ford I, Cotter MA, Cameron NE, et al. (2001) The effects of treatment with a-lipoic acid or evening primrose oil on xenobiotic toxic compounds. In this context, it has been vascular hemostatic and lipid risk factors, blood ﬂow, and well demonstrated that compounds having the propensity to peripheral nerve conduction in the streptozotocin-diabetic increase the activity and gene expression of enzymes involved rat. Metabolism 50, 868–875. in drug metabolism exhibit chemopreventive effects. In fact, 12. Huong DT & Ide T (2008) Dietary lipoic acid-dependent

some studies have indicated that a-lipoic acid is effective changes in the activity and mRNA levels of hepatic lipogenic . IP address: against tumour development(73). Evaluation of the physio- enzymes in rats. Br J Nutr 100, 79–87. logical effect of a-lipoic acid in pathological conditions 13. Ide T, Azechi A, Suzuki N, et al. (2013) Effects of dietary a-lipoic acid enantiomers on hepatic fatty acid metabolism would clarify the physiological signiﬁcance of the 170.106.202.226 in rats. J Funct Foods 5, 71–79. up-regulation of the gene expression of proteins involved in 14. Ide T, Azechi A, Kitade S, et al. (2013) Combined effect of drug metabolism and antioxidation by a-lipoic acid. This sesamin and a-lipoic acid on hepatic fatty acid metabolism needs to be clariﬁed in future studies. in rats. Eur J Nutr 52, 1015–1027.

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thesis of the microsomal drug-metabolizing enzyme system 28 Sep 2021 at 21:50:59 and its relationship to the proliferation of endoplasmic mem- Acknowledgements branes. A morphological and biochemical study. J Cell Biol The present study was supported by a grant-in-aid for scienti- 25, 627–639. 16. Jia Z, Hallur S, Zhu H, et al. (2008) Potent upregulation of ﬁc research (Scientiﬁc Research C, no. 22580143) from the glutathione and NAD(P)H:quinone oxidoreductase 1 by Japan Society for the Promotion of Science and a grant from alpha-lipoic acid in human neuroblastoma SH-SY5Y cells:

the Ministry of Agriculture, Forestry, and Fisheries research protection against neurotoxicant-elicited cytotoxicity. Neuro- , subject to the Cambridge Core terms of use, available at project ‘Development of evaluation and management methods chem Res 33, 790–800. for supply of safe, reliable and functional food and farm 17. Elangovan S & Hsieh TC (2008) Control of cellular redox produce’. status and upregulation of quinone reductase NQO1 via The author’s contributions are as follows: T. I. designed and Nrf2 activation by a-lipoic acid in human leukemia HL-60 cells. Int J Oncol 33, 833–838. carried out the animal experiment, analysed the data and 18. Ki SH & Kim SG (2008) Phase II enzyme induction by alpha- prepared the manuscript. lipoic acid through phosphatidylinositol 3-kinase-dependent The author declares no conflicts of interest. C/EBPs activation. Xenobiotica 38, 587–604. 19. Odbayar TO, Kimura T, Tsushida T, et al. (2009) Isoenzyme- specific up-regulation of glutathione transferase and aldo- keto reductase mRNA expression by dietary quercetin in References rat liver. Mol Cell Biochem 325, 121–130. 1. Shay KP, Moreau RF, Smith EJ, et al. (2009) Alpha-lipoic acid 20. Reeves PG, Nielsen FH & Fahey GC Jr (1993) AIN-93 purified

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https://doi.org/10.1017/S0007114514000841