The Journal of Toxicological Sciences (J. Toxicol. Sci.) 475 Vol.45, No.8, 475-492, 2020

Original Article Peroxisome proliferator-activated α agonist-induced decarboxylase expression in the rat and mouse Yoko Amagase*, Yumiko Mizukawa* and Tetsuro Urushidani

Department of Pathophysiology, Faculty of Pharmaceutical Sciences, Doshisha Women’s College of Liberal Arts, Kodo, Kyotanabe, Kyoto, 610-0395, Japan

(Received September 4, 2019; Accepted May 15, 2020)

ABSTRACT — By analysis of the data from the Toxicogenomics Database (TG-GATEs), gene (Hdc) was identified as largely and commonly upregulated by three fibrates, clofi- brate, fenofibrate, and WY-14,643, which are known to induce hepatocellular hypertrophy and prolifera- tion via stimulation of peroxisome proliferator-activated receptor α (PPARα) in rodents. As has been reported to be involved in the proliferation of liver cells, the present study was conducted to focus on Hdc. Among other related to histidine and histamine, the expression of the gene of histamine ammonia (Hal) was exclusively mobilized by the three fibrates. The expression of Hdc, which was usually very low in the liver, was increased with the repeated administration of fibrates, and concomitant- ly, the constitutive expression of Hal was suppressed. An interpretation is that the formation of urocanic acid from histidine under the normal condition switches to the formation of histamine. The mobilization of gene expression of Hdc and Hal by PPARα agonists could not be reproduced in primary cultured hepa- tocytes. The Hdc mRNA appeared to be translated to a protein which is processed differently from but similarly to gastric mucosa. Surprisingly, the fibrates caused hepatic hypertrophy but no induction of Hdc mRNA at all in mice. These results revealed that the changes in the histidine catabolism by PPARα agonists might be partially, but not directly, involved in the hepatocyte proliferation in rats, and there is a large genetic distance even between rat and mouse.

Key words: Toxicogenomics, Transcriptome database, Species difference, Fibric acid, Histidine decarboxylase

INTRODUCTION of the key causal genes (Gonzalez and Shah, 2008). Our analysis of the data in the Toxicogenomics Data- Peroxisome proliferator-activated receptor α (PPARα) base (TG-GATEs) in the previous study (Mizukawa et al., agonists are the first-choice drugs for dyslipidemic 2020) proposed a list of genes that are reproducibly and patients with high triglyceride. Their repeated administra- markedly upregulated by PPARα agonists. We suggested tion to rodents, however, certainly causes hepatic hyper- in the study that a) some important changes may occur trophy within a few days and thereafter hepatic tumor in before the elevation of c-Myc, b) various genes, which a high incidence following long-term administration. This are scarcely related to lipid , are upregulat- toxicity has been considered rodent-specific and therefore ed together with that related to lipid metabolism and to considered to be no risk in humans (Desvergne and Wahli, proliferation, c) many of the genes unrelated to lip- 1999; Han et al., 2017). Hepatic hypertrophy as well as id metabolism are not induced in the primary cultured tumor has been attributed to PPARα by analysis of knock- hepatocytes, and d) the gene list might contain genes that out mice (Lee et al., 1995) and thus the species differ- are involved in the rodent-specific . Among ence occurs in the reaction after the step of PPARα acti- them, we pay attention to histidine decarboxylase gene vation, but its detailed mechanism is unknown, although (Hdc), which was highly upregulated by PPARα agonists the upregulation of c-Myc has been suggested to be one and is physiologically interesting.

Correspondence: Tetsuro Urushidani (E-mail: [email protected]) *These authors equally contributed to this work.

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Histidine decarboxylase (HDC) is the only together with the other drugs, middle dose data were used synthesizing histamine, and is usually quite low in adult instead. During the project, it sometimes occurred that tissues except mast cells, gastric ECL cells, or , gene expression changes in single dose were so small that where HDC is constitutively expressed and histamine is even the highest dose showed almost the same results as stored in the secretory vesicles (Ichikawa et al., 2010). control vehicle, and then additional experiments were per- HDC is enriched in some developing organs, and the liv- formed employing higher dose(s). In two cases (CPZ and er is the most enriched organ in the rat fetus (Taguchi et ADP) among them, the low dose was omitted and indi- al., 1984). HDC activity is markedly induced in the liv- cated as NA in the table. They are basically aligned as the er, , or spleen under pathophysiological conditions order of the project number but the drugs with high ago- where various are involved (Endo et al., 1986). nistic potency of PPARα are dispersed in order to avoid Histamine produced by induced HDC is reported to play the overlapping of the symbols on the 3D-graph. a role in cell proliferation, such as in vascular smooth The concentrations of the drugs for primary cultured muscle cells during restenosis of coronary artery (Fang et hepatocytes were independently determined by a pilot test al., 2005) and various cancer types (Blaya et al., 2010; for cellular toxicity. The highest concentration was set to Medina and Rivera, 2010). It is also reported that HDC 10–20% of the lethal concentration as estimated by lac- is induced in the liver by partial hepatectomy (Ishikawa tate dehydrogenase leakage over 24 hr. When the cells et al., 1970; Chang et al., 2010), which markedly stimu- could tolerate as much as 10 mM or the level equal to lates hepatic cell proliferation. Consequently, we consider the solubility limit of the compound in DMSO (allowed it to be highly possible that induction of Hdc is involved to add up to 0.1% in the final concentration), the highest in hepatic hypertrophy caused by PPARα agonists. concentration was set to either value. In general, the max- imum concentration (high) was decreased by a factor of 5 MATERIALS AND METHODS to middle and low concentration (Table 1). The precise protocols for TG-GATEs are also availa- Gene expression data in the database ble from the same web page. In general, for in vivo pro- Transcriptome data of 132 chemicals, body weight and tocol, 7-week-old male Sprague Dawley (SD) rats were liver weight changes were obtained from the database, treated with three doses of each test drug (low, middle, TG-GATEs (http://toxico.nibiohn.go.jp/) (Urushidani, high, and vehicle: 5 rats each) and sacrificed 3, 6, 9, and 2008). The doses, the vehicles, and the abbreviations for 24 hr after single oral dosing as well as 24 hr after repeat- the 132 drugs analyzed in the present study are summa- ed dosing for 3, 7, 14, and 28 days. Toxicology data were rized in Table 1. Most of the drugs were dissolved or sus- obtained from 5 animals while gene expression analysis pended in 0.5% methylcellulose (MC) or corn oil and by Affymetrix GeneChip (Rat Expression Array 230 2.0) administered orally. In some cases indicated as “saline” in was performed for 3 out of 5. As for in vitro protocol, iso- the column for vehicle, the drug was dosed by intravenous lated hepatocytes from 7 weeks SD rats according to the injection. was diluted with distilled water. The standard method were cultivated with a collagen coated highest dose of each drug for repeated administration was six-well plate for 24 hr, and 2, 8, and 24 hr after the addi- first determined by a small-scale dose finding test (repeat- tion of the test drug (low, middle, high, and vehicle: 2 ed administration for 1 week) such that the expected sur- wells each) and the cells were harvested and analyzed for vival of the animals until the end of 28 days of repeated gene expression (Tamura et al., 2006). administration was set as the first priority. In general, the The digital image files were processed by Affymetrix highest dose was reduced by the factor of the square root Microarray Suite version 5.0 and the intensities were nor- of 10, to middle and to low dose, and the same dose lev- malized for each chip by setting the mean intensity to 500 el was employed for the single dosing. When the toxicity (per chip normalization). was expected to be highly increased in repeated adminis- tration in advance, like in anti-inflammatory, anti-cancer, Additional experiments in the present study or immunosuppressive drugs, higher dose levels were For the data newly added by the present study, male employed for single dose experiments such that at least 6-week-old SD rats and C57BL/6N mice were purchased one dose level was common with the repeated dosing. In from Shimizu Laboratory Supplies Co. and used after one some cases, because of the death or early sacrifice when week of acclimatization. The experimental protocol was moribund, the animals for high dose at 28 days were approved by The Animal Research Committee in Doshi- lacking and this is indicated with parentheses in Table 1. sha Women’s College of Liberal Arts. When the high dose data of these drugs were presented In order to see the reproducibility of the data in TG-

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Table 1. List of the drugs stored in “TG-GATEs”, and their abbreviations, doses, and vehicles. vehicle single dose (mg/kg) repeated dose (mg/kg) concentration in vitro (µM) Drugs Abbrevation in vivo low middle high low middle high low middle high WY-14,643 WY corn oil 10 30 100 10 30 100 8 40 200 acetaminophen APAP 0.5% MC 300 600 1000 300 600 1000 1000 3000 10000 INAH 0.5% MC 200 600 2000 50 100 200 400 2000 10000 PB 0.5% MC 100 150 300 10 30 100 1000 3000 10000 valproic acid VPA 0.5% MC 45 150 450 45 150 450 400 2000 10000 acetylsalicylic acid ASA 0.5% MC 450 1000 2000 45 150 450 120 600 3000 RIF 0.5% MC 20 60 200 20 60 200 2.8 14 70 naphthyl isothiocyanate ANIT corn oil 15 50 150 1.5 5 15 8 40 200 hexachlorobenzene HCB corn oil 300 1000 2000 30 100 300 0.6 3 15 allyl alcohol AA corn oil 3 10 30 3 10 30 0.8 4 20 phenylbutazone PhB 0.5% MC 20 60 200 20 60 200 16 80 400 diclofenac sodium DFNa 0.5% MC 10 30 100 1 3 10 16 80 400 omeprazole OPZ 0.5% MC 100 300 1000 100 300 1000 4.8 24 120 ethionine ET 0.5% MC 25 80 250 25 80 250 400 2000 10000 indomethacin IM 0.5% MC 5 15 50 0.5 1.6 5 12 60 300 CPZ 0.5% MC NA 45 150 4.5 15 45 0.8 4 20 thioacetamide TAA 0.5% MC 4.5 15 45 4.5 15 45 12 60 300 carbamazepine CBZ 0.5% MC 30 100 300 30 100 300 12 60 300 nitrofurantoin NFT 0.5% MC 100 300 600 10 30 100 5 25 125 benzbromarone BBr 0.5% MC 20 60 200 20 60 200 0.6 3 15 diazepam DZP 0.5% MC 25 75 250 25 75 250 5 25 125 cyclophosphamide CPA 0.5% MC 15 50 150 1.5 5 15 8 40 200 MP 0.5% MC 10 30 100 10 30 100 0.6 3 15 phenytoin PHE 0.5% MC 600 1200 2000 60 200 600 2.4 12 6 coumarin CMA 0.5% MC 15 50 150 15 50 150 12 60 300 propylthiouracil PTU 0.5% MC 15 50 150 15 50 150 160 800 4000 allopurinol APL 0.5% MC 15 50 150 15 50 15 5.6 28 140 bromobenzene BBZ corn oil 30 100 300 30 100 300 8 40 200 sulfasalazine SS 0.5% MC 100 300 1000 100 300 1000 4 20 100 CIM 0.5% MC 100 300 1000 100 300 1000 12 60 300 haloperidol HPL 0.5% MC 3 10 30 3 10 30 2 10 50 amiodarone AM 0.5% MC 200 600 2000 20 60 200 0.28 1.4 7 FP 0.5% MC 2 6 20 2 6 20 1.2 6 30 TRZ 0.5% MC 10 30 100 10 30 100 0.4 2 10 adapin ADP 0.5% MC NA 100 300 10 30 100 3 15 75 labetalol LBT 0.5% MC 45 150 450 45 150 450 5.6 28 140 methyltestosterone MTS 0.5% MC 30 100 300 30 100 300 1.6 8 40 glibenclamide GBC corn oil 100 300 1000 100 300 100 2.4 12 60 griseofulvin GF corn oil 100 300 1000 100 300 1000 1.2 6 30 flutamide FT corn oil 15 50 150 15 50 150 3 15 75 perhexiline PH 0.5% MC 15 50 150 15 50 150 0.4 2 10 azathioprine AZP 0.5% MC 3 10 30 3 10 30 0.14 0.72 3.6 ketoconazole KC 0.5% MC 10 30 100 10 30 100 0.6 3 15 tetracycline TC 0.5% MC 100 300 1000 100 300 1000 1 5 25 lomustine LS 0.5% MC 0.6 2 6 0.6 2 6 4.8 24 120 clofibrate CFB corn oil 30 100 300 30 100 300 12 60 300 ciprofloxacin CPX 0.5% MC 100 300 1000 100 300 1000 1 5 25 pemoline PML 0.5% MC 7.5 25 75 7.5 25 75 3 15 75 chlormezanone CMN corn oil 50 150 500 50 150 500 10 50 250 metformin MFM 0.5% MC 100 300 1000 100 300 1000 40 200 1000

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Table 1. (Continued).

vehicle single dose (mg/kg) repeated dose (mg/kg) concentration in vitro (µM) Drugs Abbrevation in vivo low middle high low middle high low middle high tamoxifen TMX corn oil 6 20 60 6 20 60 0.12 0.6 3 ethinylestradiol EE corn oil 1 3 10 1 3 10 0.6 3 15 carbon tetrachloride CCl4 corn oil 100 150 300 10 30 100 1000 3000 10000 MDP corn oil 60 200 600 60 200 600 2 10 50 methimazole MTZ 0.5% MC 10 30 100 10 30 100 400 2000 10000 monocrotaline MCT 0.5% MC 3 10 30 3 10 (30) 3.6 18 9 vitamin A VA corn oil 10 30 100 10 30 100 0.3 1.5 7.5 TAC 0.5% MC 3 10 30 3 10 30 8 40 200 moxisylyte MXS 0.5% MC 50 150 500 50 150 500 24 120 600 IPA 0.5% MC 6 20 60 6 20 60 80 400 2000 chloramphenicol CMP 0.5% MC 100 300 1000 100 300 1000 18 90 450 nitrofurazone NFZ 0.5% MC 30 100 300 10 30 100 12 60 300 IMI 0.5% MC 10 30 100 10 30 100 4 20 100 AMT 0.5% MC 15 50 150 15 50 150 2.4 12 60 HYZ 0.5% MC 10 30 100 10 30 100 6 30 150 ibuprofen IBU 0.5% MC 60 200 400 20 60 200 40 200 1000 naproxen NPX 0.5% MC 20 60 200 6 20 (60) 80 400 2000 quinidine QND 0.5% MC 20 60 200 20 60 200 8 40 200 rosiglitazone RGZ 0.5% MC 20 100 500 20 100 500 12 60 300 furosemide FUR 0.5% MC 30 100 300 30 100 300 100 500 2500 chlorpropamide CPP 0.5% MC 30 100 300 30 100 300 30 150 750 nicotinic acid NIC 0.5% MC 100 300 1000 100 300 1000 400 2000 10000 erythromycin ethylsuccinate EME 0.5% MC 100 300 1000 100 300 1000 3 15 75 EBU 0.5% MC 100 300 1000 100 300 1000 160 800 4000 mefenamic acid MEF 0.5% MC 30 100 300 30 100 300 6 30 150 FAM 0.5% MC 100 300 1000 100 300 1000 28 140 700 RAN 0.5% MC 100 300 1000 100 300 1000 160 800 4000 chlorpheniramine CHL 0.5% MC 3 10 30 3 10 30 8 40 200 nifedipine NIF 0.5% MC 100 300 1000 100 300 1000 10 50 250 diltiazem DIL 0.5% MC 80 240 800 80 240 800 10 50 250 tannic acid TAN 0.5% MC 100 300 1000 100 300 1000 0.4 2 10 captopril CAP 0.5% MC 100 300 1000 100 300 1000 400 2000 10000 enalapril ENA 0.5% MC 60 200 600 60 200 600 80 400 2000 theophylline TEO 0.5% MC 20 60 200 20 60 200 400 2000 10000 caffeine CAF 0.5% MC 10 30 100 10 30 100 400 2000 10000 papaverine PAP 0.5% MC 40 120 400 40 120 400 4 20 100 penicillamine PEN 0.5% MC 100 300 1000 100 300 1000 400 2000 10000 fenofibrate FFB 0.5% MC 10 100 1000 10 100 1000 1.2 6 30 sulindac SUL 0.5% MC 15 50 150 5 15 50 80 400 2000 triamterene TRI 0.5% MC 15 50 150 15 50 150 1.2 6 30 disopyramide DIS 0.5% MC 40 120 400 40 120 400 100 500 2500 meloxicam MLX 0.5% MC 10 30 100 3 10 30 2 10 50 mexiletine MEX 0.5% MC 40 120 400 40 120 400 0.6 3 15 tiopronin TIO 0.5% MC 100 300 1000 100 300 1000 1 5 25 acetazolamide ACZ 0.5% MC 60 200 600 60 200 600 24 120 600 DSF 0.5% MC 60 200 600 60 200 600 2.4 12 60 PMZ 0.5% MC 20 60 200 20 60 200 3.2 16 80 bendazac BDZ 0.5% MC 100 300 1000 30 100 300 8 40 200 colchicine COL 0.5% MC 1.5 5 15 0.5 1.5 5 200 1000 5000 tolbutamide TLB 0.5% MC 100 300 1000 100 300 1000 80 400 2000

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Table 1. (Continued).

vehicle single dose (mg/kg) repeated dose (mg/kg) concentration in vitro (µM) Drugs Abbrevation in vivo low middle high low middle high low middle high sulpiride SLP 0.5% MC 300 1000 2000 100 300 1000 200 1000 5000 acarbose ACA 0.5% MC 100 300 1000 100 300 1000 400 2000 10000 simvastatin SST 0.5% MC 40 120 400 40 120 400 2.4 12 60 ajmaline AJM 0.5% MC 30 100 300 30 100 300 12 60 300 dantrolene DTL 0.5% MC 25 75 250 25 75 250 0.4 2 10 triazolam TZM 0.5% MC 100 300 1000 100 300 1000 0.4 2 10 CPM 0.5% MC 10 30 100 10 30 100 1.6 8 40 trimethadione TMD 0.5% MC 50 150 500 50 150 500 400 2000 10000 telbinafine TBF 0.5% MC 75 250 750 75 250 750 0.6 3 15 chlormadinone CLM 0.5% MC 300 1000 2000 100 300 1000 1.6 8 40 danazol DNZ 0.5% MC 300 1000 2000 100 300 1000 1.4 7 35 benziodarone BZD 0.5% MC 30 100 300 30 100 300 1 5 25 etoposide ETP 0.5% MC 10 100 1000 3 10 30 14 70 350 cisplatin CSP saline 0.3 1 3 0.1 0.3 1 8 40 200 lornoxicam LNX 0.5% MC 1 3 10 0.3 1 3 0.6 3 15 carboplatin CBP saline 10 30 100 1 3 10 120 600 3000 bromoethylamine BEA saline 6 20 60 2 6 20 20 100 500 ethionamide ETH 0.5% MC 100 300 1000 30 100 (300) 24 120 600 nimesulide NIM 0.5% MC 30 100 300 10 30 100 3 15 75 ethanol ETN water 400 1200 4000 400 1200 4000 400 2000 10000 phenacetin PCT 0.5% MC 300 1000 2000 100 300 1000 24 120 600 bucetin BCT 0.5% MC 300 1000 2000 100 300 1000 12 60 300 phenylanthranilic acid NPAA 0.5% MC 300 1000 2000 100 300 1000 8 40 200 cephalothin CLT saline 300 1000 2000 300 1000 2000 120 600 3000 cyclosporine A CSA corn oil 30 100 300 10 30 100 0.24 1.2 6 doxorubicin DOX saline 1 3 10 0.1 0.3 1 0.08 0.4 2 puromycin aminonucleoside PAN saline 12 40 120 4 12 40 100 500 2500 acetamidofluorene AAF 0.5% MC 100 300 1000 30 100 300 2 10 50 nitrosodiethylamine DEN 0.5% MC 10 30 100 3 10 30 400 2000 10000 ticlopidine TCP 0.5% MC 100 300 1000 30 100 300 2.4 12 60 gentamicin GMC saline 10 30 100 10 30 100 1.2 6 30 gemfibrozil GFZ corn oil 30 100 300 30 100 300 4 20 100 Parenthese in the high dose mean that the data of the administration for 28 days are lacking because of the death of animals, and replaced by the data of middle dose when presented.

GATEs, rats (N=3) received 1000 mg/kg of FFB or was performed using PrimeScript RT reagent kit with 30 mg/kg WY p.o. for 3 days and were sacrificed 24 hr gDNAeraser (Takara Bio) to obtain cDNA and real-time after the last dose by exsanguination under isoflurane PCR was done with SYBR premix Ex Taq II (Takara Bio) anesthesia. The control rats received vehicle (0.5% MC using Roter-Gene Q (Qiagen, Osaka, Japan). The prim- for FFB and corn oil for WY). For mice, 100 mg/kg of ers used were purchased from Takara Bio; RA058783 FFB were administered for 3 and 7 days (N=4 each), or for rat Hdc and MA131065 for mouse Hdc. For a refer- 100 mg/kg of WY for 3 days (N=3), and the mice were ence gene, β-actin (RA015375 for rat and MA050368 for sacrificed by exsanguination under isoflurane anesthesia. mouse) was employed and the results were expressed as For real-time PCR, a part of the liver was soaked in RNA the ratio to β-actin. later and the remaining part was frozen by liquid nitrogen The sample for protein analysis was homogenized in for protein analysis. 4 volumes of a homogenizing buffer (113 mM mannitol, The sample for PCR was homogenized and the total 37 mM sucrose, 5 mM PIPES-Tris, 0.4 mM EDTA, RNA was extracted using NucleoSpinRNA kit (Takara pH=6.7, supplemented with protein inhibitor cocktail) Bio, Shiga, Japan). Reverse transcription reaction with a sonicator (Vibra Cell, Sonics & Materials Inc.,

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Newtown, CT, USA) for 30 sec x 4 on ice, and centri- dilution in TPBS of Alexa Fluor 488 anti-rabbit IgG fuged at 500 x g for 10 min to remove debris. The super- (Sigma Aldrich Japan) at room temperature for 2 hr. natant was centrifuged at 100,000 x g for 1 hr (Optima Immunofluorescence was observed by using EOS FL TLX, Beckman Coulter, Tokyo, Japan), and the superna- (Thermo Fischer Scientific, Tokyo, Japan) with constant tant was taken as a cytosol fraction and the pellet was sus- settings throughout the observation of the sections. pended in the same buffer and taken as a membrane frac- tion. For the standard of HDC, gastric mucosa and whole Data presentation and statistical analysis brain were taken from the rat and mouse, and subjected to Data are presented as means + SEM. When one pair the same protocol as above to get cytosol and membrane was compared, Student’s t-test was employed. When mul- fraction. tiple comparison against one control was made, Dunnett’s Protein samples were solubilized in a buffer (2% SDS, test was used. A difference of p < 0.05 was considered 65 mM dithiothreitol, 0.25 mM EDTA, 10% glycerol, statistically significant. 15 mM Tris, pH=6.8) and subjected to 10% SDS-poly- acrylamide electrophoresis according to standard pro- RESULTS tocol. After the electrophoresis, the proteins were trans- ferred to PVDF membrane, blocked with 5% skim milk Induction of Hdc expression by PPARα agonists in 0.5% Tween 20-PBS (TPBS), and incubated with in rat liver 1/100 diluted primary antibody (rabbit polyclonal anti- From the database, TG-GATEs, we extracted genes HDC) in blocking solution at 4°C, overnight. The mem- that were commonly and largely upregulated by three branes were washed 3 times with TPBS and incubat- fibrates, CFB, FFB, and WY, at 24 hr after the single dos- ed with 1/10,000 diluted secondary antibody (Donkey ing, and sorted them by their induction rates (Mizukawa polyclonal HRP-anti-rabbit IgG, Sigma Aldrich Japan, et al., 2020). Of these, the 4th ranking was Hdc mRNA Tokyo, Japan) in TPBS at room temperature for 2 hr. After detected by the probe set, 1370491_a_at. Because of the 3 times washing with TPBS secondary antibody was visu- scarce expression in the vehicle control, the induction alized by Western Lightning ECL Pro (Perkin-Elmer Japan, rates were so high that 67.6, 74.1, and 54.7 fold by CFB, Yokohama, Japan) and observed by an illuminator FFB, and WY, respectively. Figs. 1A-C show upregula- (C-DiGit, LI-COR). We used two commercially availa- tion of Hdc mRNA by these three agonists at all doses ble antibodies recognizing both rat and mouse HDC. One and time points. It is obvious from the figures that very (Ab-A) was from Abcam plc (Cambridge, UK) and it was low expression of Hdc was observed at any points in the against near the C terminal 20 amino acids (620 - 639) of liver of control rats, suggesting no circadian or feeding- HDC (662 amino acids in total). The other (Ab-B) was related changes occurred. In fact, all the data from con- from Atlas Antibodies (Bromma, Sweden) and it was trol rats showed “absent call” by GeneChip. In the groups raised against near the N-terminal 132 amino acids (89 - receiving PPARα agonists, upregulation of Hdc expres- 220) of HDC. sion increased dose- and time-dependently after sin- Another pair of rats receiving 30 mg/kg WY or its gle administrations. In the case of CFB, further increase vehicle control for 3 days were subjected to histo- did not occur by repeated administrations, whereas the logical analysis. The rats were perfused via left ven- expression kept increasing by repeated dosing both in tricle with PBS followed by 1% paraformaldehyde FFB and WY. under isoflurane anesthesia. The fixed liver was sub- In order to see the relationship between Hdc induc- jected to paraffin sections (Kyoto Institute of Nutrition tion and hypertrophy of the liver, data of body and liv- & Pathology, Inc., Kyoto, Japan). The sections were er weight were harvested from the database and the rel- deparaffinized and rehydrated. In the preliminary exper- ative liver weight per body weight (%) was calculated as iments, it was found that the antigenicity to both Ab-A shown in Figs. 1D-F. The enlargement of the liver start- and Ab-B was lost by the fixation and embedding, so ed as early as 24 hr after a single dose and became obvi- antigen retrieval was performed by heating the sec- ous after 3 successive doses or more in all three fibrates. tions soaked in 10 mM citrate buffer (pH=6.0) for These changes are roughly in parallel with that of Hdc 12 min by a microwave using a pressure cooker. The sec- expression. tions were blocked with 5% skim milk in TPBS for 1 hr Data of primary cultured hepatocytes are also availa- at room temperature, they were incubated with 1/50 dilu- ble to compare with in vivo data. However, all the expres- tion in the blocking solution of Ab-A at 4°C overnight, sion values in both control and treated groups were very washed 3 times with TPBS and then incubated with 1/100 low (< 70) with the judgment of “absent call” (data not

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Fig. 1. Effects of CFB, FFB, and WY on the expression of Hdc in rat liver (A-C) and on the relative liver weight per body weight (%) (D-F). : vehicle control,: low dose,: middle dose,: high dose. The data are expressed as mean + SEM, N=3 (Hdc) or N=5 (relative liver weight). *Significantly different from control, at p < 0.05 by Dunnett’s test. shown). It is obvious that fibrates lose their ability to HDC. The mRNA of Hdc is detected by 1370491_a_at. induce Hdc mRNA in the cultured hepatocytes. e) Histamine is inactivated by histamine N-methyl- (HNMT) and (DAO). The Effects of PPARα agonists on the expression of expression of HNMT gene (Hnmt) can be detected by the genes related to histidine and histamine 1385762_at and 1387382_at., and that of DAO gene The following are involved in the metabo- (Dao) can be detected by 1369491_at. lism of histamine or its precursor, histidine (Chang et al., The first probe set (1368551_at) for Prps2 appeared 2010), and their data were extracted from the database. to be low efficient and all the data showed “absent call”. a) D-ribose 5- is phosphorylated by phos- The latter two sets (1375932_at and 1398262_at) detected phoribosyl pyrophosphate synthetase 2 to form 5-phos- the expression but no drug-related changes were observed phoribosyl-1-pyrophosphate. The mRNA of this gene (data not shown). The expression of Ppat detected by (Prps2) can be detected by the probe sets 1368551_at, 1369785_at showed circadian changes in the control 1375932_at, and 1398262_at in GeneChip. group receiving vehicle, but the fibrates failed to affect b) Phosphoribosyl pyrophosphate aminotransferase them (data not shown). transfers α-amino group of glutamine to 5-phosphoribo- Figures 2A-C show the expression changes of Hal by syl-1-pyrophosphate to form histidine. The mRNA of this the three fibrates. Although not obvious in the single dos- gene (Ppat) can be detected by 1369785_at. ing, its basal expression was markedly suppressed by the c) Histidine is broken down into urocanic acid by his- repeated administration of the fibrates for 3 days and long- tidine ammonia lyase (HAL). The mRNA of this gene er. The changes in Hal expression were not observed for (Hal) can be detected by 1387307_at. in vitro either, same as the case of Hdc (data now shown). d) Histidine is alternatively turned into histamine by In contrast to histidine metabolism, histamine metabo-

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Fig. 2. Effects of CFB, FFB, and WY on the expression of Hal in rat liver. : vehicle control, : low dose, : middle dose, : high dose. The data are expressed as mean + SEM, N=3. *Significantly different from control, at p < 0.05 by Dunnett’s test.

As for other histamine-related genes, probe sets are designed for four types of receptor, i.e., 1369855_at and

1370338_at for H1, 1369914_at for H2, 1368451_at and

1388080_a_at for H3, and 1387590_at for H4. However, they were all very small with “absent call” at every dose and time point.

Specificity ofHdc expression changes It is known that the expression change of a certain gene affects the expression of its neighbor gene on the genome with various mechanisms. According to com- prehensive analysis of the interrelationship among neigh- boring gene expression (Sémon and Duret, 2006), sig- nificant interaction is mostly limited to two genes next Fig. 3. Genomic localization of Hdc and its neighboring genes to each other. We then examined the genes right next to in rat and mouse. A: Rat Hdc locates in chromosome Hdc (Fig 3). Rat Hdc locates in chromosome 3, and GA 3 between Atp8b4 and Usp8 (from 112,609,782 to 112,804,623). B: Mouse Hdc locates in chromosome binding protein transcription factor, beta subunit 1 (Gab- 2 also between Atp8b4 and Usp8 (from 126,500,669 pb1) and ATPase phospholipid transporting 8B4 (Atp8b4) to 126,707,261) which is a little longer than that of rat. exist immediately upstream and downstream, respec- Note that directly downstream of Hdc is Atp8b4 and tively, on the same strand. Slc27a2 is the closest to Hdc the “PROMPT” position is Usp8 in the rat genome, and ubiquitin specific peptidase 8 (Usp8) locates in the whereas predicted genes (Gm26697 and Gm27003, respectively) locate between them in mouse genome. position of so-called “promoter upstream transcription” The scale bar indicates the length for 100 kilobase. (Lloret-Llinares et al., 2016). Of these, expression of nei- ther Gabpb1 nor Usp8 was changed by the fibrates at any dose or time point (data not shown). Atp8b4 was judged lism did not appear to be affected by fibrates. The expres- as “absent call” by GeneChip at most of the points and sion of Hnmt showed a very low value with “absent call” no consistent results were obtained. Fig. 3 also shows the at every dose and time point by both probe sets. The genomic location in mouse genes and this is used in the expression of Dao was found to be quite high reflecting discussion. the fact that the liver is the main organ for deaminating As shown in Figs. 4A-C, Slc27a2 was the only gene histamine (Blaya et al., 2010), but it was not affected by among them upregulated by the fibrates. SLC27A2 is the administration of fibrates at all. These were observed not a simple transporter but converts free long-chain fat- in vitro as well as in vitro (data not shown). ty acids into fatty acyl-CoA esters, which thereby plays

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Fig. 4. Effects of CFB (A), FFB (B), and WY (C) on the expression of Slc27a2 in rat liver. : vehicle control, : low dose, : middle dose, : high dose. The data are expressed as mean + SEM, N=3. *Significantly different from control, at p < 0.05 by Dunnett’s test.

Fig. 5. Effects of CFB (A), FFB (B), and WY (C) on the expression of Slc27a2 in rat primary cultured hepatocytes. The values are the mean of duplicate measurements for each dose and time point. : vehicle control (DMSO), : low, : middle, : high concentration. a key role in lipid metabolism and is actually known to time point are indicated on the graph. In addition, expres- be induced by PPARα agonists (Rakhshandehroo et al., sion of acyl-CoA thioesterase 1(Acot1), which is one of 2010). This gene was highly expressed in the control and the most inducible genes by PPARα agonist in rats, is the three agonists further increased throughout the repeat- shown in Fig. 6B in the same manner as for Hdc except ed dosing period. the drugs that induced more than 1,000 at any time points Effects of the fibrates on the expression of Slc27a2 were indicated. The bar graphs for Hdc and Acot1 are were also confirmed in vitro. As shown in Fig. 5, no effect quite similar for the specified drugs. It is obvious that four of the fibrates was observed until 6 hr after their exposure, fibrates in the database, i.e., CFB, FFB, WY and GFZ, all whereas the induction effect of the drug emerged at 24 hr showed marked induction of both Hdc and Acot1. Fur- as the basal expression in the control cells decreased with thermore, BBr, AM, SST, and BZD, which were report- time, resulting in a 2.5 to 3 fold increase. ed to possess PPARα agonistic activity (Kunishima et In order to examine if the induction of Hdc is specific al., 2007; Seo et al., 2008) induced Hdc as well as Acot1. for PPARα stimulation, expression of Hdc was examined Non-steroidal anti-inflammatory drugs, which are known for 132 chemicals available in the database and shown to contain PPARα agonists (Lehmann et al., 1997), ASA, in Fig. 6A, where the data were narrowed down to con- IBU, MLX, BDZ, and LNX upregulated Hdc, where- trol and high dose (the data of middle dose was employed as ASA, BDZ, and NPAA induced Acot1 gene, but oth- when lethal cases occurred at high dose) at 24 hr after sin- ers such as DFNa, IM, NPX, MEF, SUL, and NIM did gle dosing and repeated dosing for 3, 7, 14 and 28 days, not affect either gene expression. We could not find sup- and the name of drugs that induced more than 300 at any porting reports for CMP, EBU, and TBF as PPARα ago-

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Fig. 6. Effects of 132 drugs in the database on Hdc (A) and Acot1 (B) expression in rat liver. Data of control and high dose 24 hr after single and repeated administration for 3, 7, 14, and 28 days are shown. The names of drugs that showed a value more than 300 (for Hdc) or 1000 (for Acot1) at any point are indicated on the graph. The drugs are aligned as the order in Table 1. nists, but they appeared to stimulate PPARα since they sponding column for the drug name is shaded. Of these clearly stimulated both Acot1 and Hdc. Repeated dosing 20 drugs, 16 are ranked in the upper half of the list, sug- of MP, CCl4, or ETH, and single dosing of AAF, which gesting a certain relationship between Hdc induction and all appear to be PPARα agonists in respect to the induc- Hal inhibition. Although most of the drugs that induced tion of Acot1, failed to induce Hdc, whereas repeated dos- Hdc inhibited Hal, there are many drugs inhibiting Hal ing of HCB, MDP, and DOX induced Hdc but not Acot1. without inducing Hdc. Hepatic hypertrophy tends to In summary, the drugs stimulating Acot1 but not Hdc occur where Hal expression is low (in the higher rank of expression were NPAA, MP, CCL4, ETH, and AAF, and Table 2), while atrophy tends to occur where it is that stimulating Hdc but not Acot1 expression were IBU, unchanged or high, but there are too many exceptions. MLX, LNX, HCB, MDP, and DOX. A simple explanation The expression changes of Hal in vitro were reviewed would be that the former drugs are PPARα agonists with a as well. As stated above, the fibrates showed no effect on direct inhibitory activity on Hdc expression, and the latter Hal expression up to 24 hr treatment. Among 132 drugs drugs have an activity of inducing Hdc expression inde- with any effect on Hal expression in vitro, the largest pendent on PPAR. Although there are some exceptions as decrease was observed for COL (Fig. 7A) and the larg- above, Figs. 6A and 6B are quite similar as a whole, indi- est increase was by DOX (Fig 7B). In Table 2, COL also cating that both Hdc and Acot1 are induced by the same showed a strong inhibition at 24 hr in vivo, whereas DOX mechanism, i.e., stimulation of PPARα. did not show any effect on Hal expression, showing an Next, expression of Hal, which was downregulated by apparent discrepancy between in vivo and vitro. In order the three fibrates in vivo, was examined for 132 chemi- to see the correlation between in vivo and vitro, values cals. As the inhibitory effect is difficult to see in a graph- of ratio to control at 24 hr after high dose are shown as ic presentation like Fig. 6, the results are summarized as a scatter plot (Fig 7C). It is obvious from the figure that Table 2, where the values of ratio to control of high dose there is no correlation between in vivo and in vitro. Effects (middle dose was employed when lethal cases occurred) of the expression changes in the neighboring genes could at 24 hr after single dosing and repeated dosing for 3, be excluded since there are no PPARα–responsive genes 7, 14 and 28 days are sorted by the value of 28 days. In around Hal on the rat genome. order to see possible relationship between Hal expression and hepatic hypertrophy, the data of relative liver weight Confirmation of PPARα agonist-inducedHdc per body weight were extracted from the toxicological expression by PCR and HDC protein expression reports in the database, and the point where the relative by western blotting liver weight significantly increased is shaded, and where Figures 8A, B show the confirmation of data from the it significant decreased is black-and-white inverted, in database by real-time PCR. As the sensitivity of PCR the corresponding column of Table 2. For the 20 drugs is higher than GeneChip, Hdc expression was low but with Hdc-inducing activity named in Fig. 6A, the corre- detectable in the control rats. Both FFB 1000 mg/kg (A)

Vol. 45 No. 8 Table 2. Effects of 132 drugs on the expression ofHal mRNA and on the relative liver weight per body weight in rats. 24hr 3d 7d 14d 28d 24hr 3d 7d 14d 28d 24hr 3d 7d 14d 28d MP 0.602 0.797 0.112 0.065 0.037 SST 0.717 0.487 0.668 0.960 0.692 TC 0.743 0.867 0.792 0.953 0.905 WY 0.843 0.259 0.234 0.148 0.093 CFB 0.801 0.598 0.460 0.680 0.693 EE 0.881 1.064 1.062 1.285 0.906 MCT 0.953 1.031 0.380 0.220 0.165 CMN 0.640 0.664 0.581 0.532 0.695 ETN 0.859 0.780 1.060 0.858 0.909 FFB 0.569 0.207 0.283 0.149 0.196 BBZ 0.653 0.588 0.805 1.011 0.698 TRI 0.918 1.089 1.484 0.803 0.910 OPZ 0.736 0.382 0.506 0.212 0.220 TEO 1.180 0.982 1.203 0.818 0.701 SLP 1.197 0.929 0.956 1.076 0.917 AAF 1.069 0.691 0.695 0.367 0.280 PhB 0.692 0.496 0.587 0.534 0.701 TAC 0.841 0.839 0.874 0.891 0.919 ANIT 0.681 0.571 0.368 0.297 0.288 CMP 0.889 0.856 1.075 1.033 0.710 NPX 0.712 0.476 0.532 0.397 0.944 PMZ 0.759 0.665 0.727 0.437 0.313 APAP 0.706 0.722 0.646 0.549 0.712 MXS 1.194 0.886 1.071 0.767 0.951 LS 1.203 1.085 0.738 0.474 0.353 CPP 1.093 0.795 0.766 0.677 0.720 CPA 1.042 1.164 1.199 0.779 0.952 AM 0.616 0.645 0.701 0.541 0.355 RIF 0.669 0.908 0.802 0.790 0.723 LNX 0.662 0.638 0.502 0.577 0.954 GFZ 0.623 0.375 0.465 0.304 0.368 QND 1.024 1.026 0.798 0.748 0.737 VPA 0.748 1.122 0.833 0.771 0.954 PPARα-induced HDCgeneexpressionintheratandmouseliver TCP 0.482 0.379 0.466 0.481 0.371 NIF 0.246 0.340 0.592 0.828 0.756 DEN 1.084 0.891 0.690 1.022 0.954 SUL 0.563 0.576 0.607 0.551 0.372 PHE 0.623 0.644 0.598 0.755 0.757 MFM 0.733 0.983 1.019 1.037 0.960 CCl4 0.975 0.620 0.999 0.711 0.399 BCT 0.972 0.608 0.700 0.877 0.759 CLM 0.817 0.917 0.775 0.885 0.960 AMT 0.670 0.945 0.606 0.569 0.412 BZD 0.838 0.507 0.577 0.636 0.764 ADP 0.622 0.935 0.857 1.033 0.967 DOX 0.921 0.736 0.836 0.875 0.418 ETH 0.771 0.503 0.993 0.507 0.775 SS 0.821 0.901 0.826 0.788 0.967 HYZ 0.630 0.560 0.527 0.456 0.418 MEX 0.957 0.872 0.856 0.740 0.778 MLX 0.541 0.462 0.472 0.520 0.969 HCB 1.116 0.567 0.484 0.313 0.450 EBU 0.580 0.958 0.412 0.699 0.780 CIM 0.897 0.815 0.907 0.654 0.974 BBr 0.703 0.360 0.484 0.463 0.460 AA 0.843 0.828 0.827 0.789 0.787 ENA 1.106 0.935 1.100 1.105 0.974 DIL 0.505 0.446 0.754 0.557 0.469 NPAA 0.472 0.614 0.746 0.592 0.793 CAF 0.815 0.743 0.784 0.756 0.975 DSF 0.808 0.674 0.650 0.701 0.518 TIO 0.790 0.773 1.032 0.907 0.798 ACZ 0.759 1.031 0.984 0.861 0.979 TBF 1.011 0.505 0.405 0.435 0.557 ETP 0.760 1.008 1.038 0.869 0.800 AJM 1.113 1.196 1.127 1.049 0.984 PB 0.645 0.677 0.612 0.638 0.559 CHL 0.894 1.044 0.799 0.736 0.806 CAP 1.060 1.288 1.288 1.066 0.984 MTS 0.872 1.114 0.643 0.596 0.561 BEA 0.807 0.926 0.937 0.778 0.807 ACA 1.169 1.242 0.907 0.911 0.993 COL 0.303 0.175 0.481 0.836 0.562 TLB 0.814 0.785 0.762 0.732 0.808 RAN 1.088 1.317 1.008 1.100 0.997 CBZ 0.560 0.688 0.576 0.651 0.568 FUR 0.789 0.833 0.851 0.829 0.811 NFT 1.237 0.955 1.154 0.830 1.009 IBU 0.743 0.713 0.765 0.471 0.568 CPM 0.856 1.098 0.971 0.832 0.817 TMD 0.811 0.782 0.866 0.905 1.046 DFNa 0.291 0.900 0.887 0.936 0.575 PML 0.866 0.952 0.932 0.822 0.818 INAH 2.379 0.963 0.886 0.839 1.047 PCT 0.459 0.869 0.482 0.627 0.582 GBC 0.825 1.090 0.822 0.903 0.834 MDP 0.841 1.319 1.278 0.815 1.058 CBP 1.114 0.894 0.708 0.645 0.586 DNZ 0.752 0.705 1.016 0.823 0.836 CPZ 0.665 1.110 0.946 0.898 1.059 AZP 0.811 0.690 0.646 0.769 0.604 BDZ 0.726 0.647 0.851 0.910 0.836 TMX 1.376 1.221 1.086 0.971 1.075 DZP 1.252 1.112 0.539 0.647 0.608 GMC 0.794 0.841 1.045 0.741 0.842 NIC 1.093 1.056 0.885 0.916 1.082 DIS 0.999 0.656 0.765 0.595 0.619 ET 0.960 1.469 1.181 1.026 0.847 IPA 1.193 1.312 1.143 1.014 1.112 TAA 0.863 1.300 0.874 0.918 0.622 CSP 0.908 1.056 0.883 0.811 0.856 LBT 0.879 0.748 0.923 1.100 1.121 MEF 0.653 0.595 0.775 0.522 0.635 TRZ 1.038 0.896 0.790 1.139 0.860 APL 0.908 1.178 1.256 1.236 1.124 KC 0.745 1.084 0.834 0.885 0.645 IM 0.630 0.706 0.804 0.638 0.863 CPX 0.721 0.961 0.977 0.821 1.125 PEN 1.401 0.856 0.968 0.827 0.654 TAN 0.922 0.943 1.056 0.832 0.869 FAM 0.952 0.948 1.021 1.151 1.126 IMI 0.704 0.758 0.705 0.513 0.656 PH 0.958 1.105 0.732 0.769 0.872 TZM 1.094 0.860 1.144 1.037 1.162 RGZ 0.923 0.910 1.402 1.371 0.671 PAP 1.124 0.664 0.884 0.756 0.872 MTZ 0.622 0.876 1.050 1.349 1.193 EME 0.736 0.814 0.921 0.731 0.671 DTL 1.243 0.867 0.947 0.867 0.874 FP 0.801 1.463 1.366 0.909 1.228 CMA 0.474 0.660 0.875 0.714 0.674 CSA 1.070 1.098 1.581 0.792 0.875 HPL 0.947 1.315 1.208 1.234 1.246 NIM 0.535 0.625 0.620 0.656 0.685 VA 0.886 0.925 0.927 0.489 0.879 PAN 0.880 0.911 0.984 0.965 1.313 ASA 1.129 0.905 0.721 0.639 0.687 CLT 0.762 0.906 0.785 0.796 0.881 NFZ 0.507 0.880 0.977 1.054 1.363

Vol. 45No. 8 GF 0.975 0.596 0.950 0.754 0.689 FT 0.840 0.614 0.837 0.655 0.889 PTU 0.867 0.774 1.226 1.255 1.504 Hal expression values of ratio to control in high dose group (data of middle dose group were employed when the high dose data were lacking as shown in Table 1) at 24 hr after single dosing and repeated dosing for 3, 7, 14 and 28 days are shown. The order of the drugs was sorted by the value of 28 days such that the higher the rank, the stronger inhibitory effect on Hal expression

at the end of repeated administration. When relative liver weight per body weight was significantly (p < 0.05) increased, the corresponding column is shaded, and the column is black-and- 485 white inverted when the relative liver weight was significantly (p<0.05) decreased. For the drugs that inducedHdc as shown in Fig. 6A, the corresponding columns are shaded. 486

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Fig. 7. Effects of various drugs on the expression of Hal. A: Inhibitory effects of colchicine (COL) on the expression of Hal in cultured rat hepatocytes. COL showed the largest inhibition among the drugs in the database. : vehicle control (culture medium), : low, 200 μM, : middle, 1 mM, : high, 5 mM. B: Stimulatory effects of doxorubicin (DOX) on the expression of Hal in cultured rat hepatocytes. DOX showed the largest increment among the drugs in the database. : vehicle control (DMSO), : low, 80 nM, : middle, 400 nM, : high, 2 μM. C: Scattered plot of Hal expression in vivo and in vitro rat liver. Ratio to control was calculated at 24 hr after treatment with high concentration for 132 drugs (see Table 1), and plotted in vivo data on the ordinate and in vitro on the abscissa. Obviously no correlation is seen.

Fig. 8. Effects of FFB 1000 mg/kg (A) or WY 30 mg/kg (B) orally administered for 3 days on the expression of Hdc in the rat liver. The expression of Hdc was quantified by real-time PCR and normalized by β-actin. The data are expressed as mean + SEM (N=3). ** Significantly different from control by Student’s t-test at p < 0.01.

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Fig. 9. Detection of HDC protein in control (C1-3) and FFB 1000 mg/kg for 3 days (F1-3) treated rat liver by Ab-A antibody (against C-terminal fragment, from Abcam) as primary antibody. A: Cytosolic fraction. Clear 30 kDa bands are visible ex- clusively in the rats treated with FFB. B: Membrane fraction. When the detection sensitivity was highly increased (obvious from the over exposure of the molecular weight marker with Westernsure Pen in the middle lane), the 30 kDa bands became visible exclusively in the rats treated with FFB. This antibody recognizes 75 kDa, which is close to the full length of HDC, in brain membrane fraction (Br) but not in the gastric membrane (Ga). In F3 lane, a 65kDa band is visible. This band is always detected by the other antibody (Ab-B) both in the membrane fraction of gastric mucosa and FFB-treated liver, but usually not by Ab-A, and this is an exceptional case.

Fig. 10. Immunofluorescent detection of HDC protein in the liver from the control (A) and WY 30 mg/kg for 3 day treated rats (B). Sections of the liver were incubated with Ab-A antibody (against C-terminal fragment, from Abcam) as primary antibody and visualized by Alexa 488-anti rabbit IgG, and observed under the exact same optical condition. HDC proteins appear to be expressed in hepatic parenchymal cells, without enrichment in any specific cell types. Scale bar: 50 μm and WY 30 mg/kg (B) were administered for 3 days and species, which forms a 110 kDa dimer (Ichikawa et al., they markedly induced Hdc expression. As the control 2010). In our preliminary experiments, the optimal con- values were close to the detection limit, the value of ratio dition was determined for the antibodies obtained from to control was relatively imprecise, but the value ca. 30 Abcam (Ab-A) and Atras (Ab-B), using samples from fold is comparable to the one in the database, 50 to 70 rat brain and gastric mucosa, known to be enriched in fold. HDC. HDC protein appeared to be labile as report- Protein expression of HDC was examined by west- ed (Ichikawa et al., 2010) especially in the solubilized ern blotting using antibodies with different epitopes. condition, and it was quite difficult to obtain reproduci- It is known that HDC is translated as a 74 kDa precur- ble results regarding its fragmentation. The most reliable sor protein and post-translationally cleaved to a 54 kDa results were as follows. Ab-A recognized a 74 kDa band

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Y. Amagase et al. in the brain membrane fraction and 32 kDa band in the kg. Because of the high viscosity of the suspension for gastric cytosol fraction. Ab-B recognized a faint 74 kDa 1000 mg/kg, the physical burden of this dose was con- and a 45 kDa band in the brain membrane fraction and a sidered to be too heavy for mice. We then chose 100 mg/ 110 kDa band in the brain cytosol. Ab-B also recognized kg of FFB for the mouse study and administered it for 3 a faint 74 kDa and 65 kDa bands in the gastric membrane and 7 days. As was in the rats, the expression of Hdc was fraction but nothing in the gastric cytosol fraction. These low but detectable in the liver of control mice by PCR. are in accordance with the proposed mechanism that the Surprisingly, Hdc was never induced by FFB at all in full length (74 kDa) of HDC is cleaved by caspase 9 into spite of the fact that the hypertrophy started after 3 days 54kDa active enzyme and 20 kDa C-terminal fragment and became marked after 7 days (Figs. 11A, B). Consid- (Tanaka et al., 1998; Furuta et al., 2007), and this is con- ering the possibility of insufficiency of FFB dosage, we sistent with the epitopes of the antibodies, i.e., C-terminal employed 100 mg/kg of WY, which was higher than the for Ab-A and N-terminal for Ab-B, considering the inac- dose showing the maximal effect on both Hdc induction curacy of the molecular weight on SDS-PAGE. This indi- and hepatic hypertrophy in rats. As shown in Fig. 11C, cated that the post-translational processing of HDC is dif- D, however, WY did not elicit any inducing effects on ferent in tissues. Figs. 9A, B show the western blotting Hdc, whereas the relative liver weight per body weight analysis of the membrane and cytosol fractions of rat liv- was weakly, but significantly increased. As expected, er using Ab-A as primary antibody. In the cytosol frac- HDC proteins could not be detected by western blotting tions from FFB-treated rats (Fig. 9A), clear 32 kDa bands, in the liver of the mice treated with FFB or WY (data not which are usually present in the gastric cytosol, are visi- shown). ble, and these bands are absent from the control. When the sensitivity of the detector increased, this band became DISCUSSION visible in the membrane fraction of FFB-treated, but not in the control liver. This band could not be detected in In the normal condition, the expression of Hdc was the membrane fraction of gastric mucosa, in contrast to very low with “absent call” by GeneChip and barely cytosolic fraction (Fig. 9B). Ab-B also visualized bands detectable by real-time PCR in matured rat liver, and thus in the position similar to that of gastric mucosa exclusive- the induction rate by the fibrates was observed to be quite ly in FFB-treated rats (data now shown). large like several tens to hundred fold as in other tissues Figures 10A, B show the immunostaining of rat liv- where Hdc is pathophysiologically induced (Ichikawa et er sections. The cytosol of the hepatocytes from WY- al., 2010; Kikuchi et al., 1997). treated rats showed positive staining by Ab-A compared Overviewing other genes involving histidine and/or with control rats. No characteristic staining of vascular histamine, Hal was the only gene of which expression endothelium, bile ducts, or Kupffer cells were seen. was affected by fibrates. The induction of Hdc was obvi- ous 24 hr after a single dosing of fibrates, whereas the Experiments using mice downregulation of Hal emerged after 3 days of adminis- From the results described above, it was revealed that tration. Histidine is converted to histamine by HDC or to fibrates strongly induced Hdc mRNA that was translated urocanic acid by HAL. The present results could be inter- to HDC protein in the rat liver. One important question preted that HDC is induced to produce histamine resulting is whether HDC or histamine is involved in the pharma- in the reduction of histidine and then the pathway to pro- cology and/or toxicology of PPARα agonists. In order to duce urocanic acid is suppressed to preserve the former investigate this point, the most powerful strategy would pathway, namely, the switching of histidine metabolism be the use of HDC-, or -knockout mice occurs. that are now available (Ichikawa et al., 2010; Ohtsu et al., All the subtypes of histamine receptor (H1 to H4) could 2001; Masaki et al., 2005). Therefore, we examined if the not be quantified by GeneChip irrespective of stimula- same phenomena in rats occur also in mice, which have tion by fibrates. This does not necessarily mean that all been considered to be the same in respect to the phar- the receptors for histamine are absent in the liver, since macology and toxicology of fibrates, such as hypolipi- usually the number of the receptor is small and the turn- demic effects and hepatic hypertrophy leading to tumor over rate is low compared with enzymes, resulting in

(Desvergne and Wahli, 1999). the small amount of mRNA. In fact, α1 and β2 receptors Looking at the rat data in Fig. 1E, increase in the rela- are known to exist and play important roles in the liver tive liver weight per body weight by FFB 100 mg/kg for (Aggerbeck et al., 1983; Ghosh et al., 2012), but mostly 3 days was almost the same extent as that by 1000 mg/ their mRNAs were judged as “absent call” by GeneChip

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(data not shown). Another possibility is a putative intra- (Maronpot et al., 2010). If HDC or its product, histamine, cellular histamine that was reported to be is involved in hepatomegaly, it could play a role in cell involved in the DNA synthesis stimulated by partial hepa- proliferation, since it is unlikely that HDC protein is the tectomy (Brandes et al., 1992). However, this protein has main part of induced enzymes, and/or histamine induces not been identified (Blaya et al., 2010) and could not be many enzymes in hepatocytes. In fact, many genes relat- verified at present. ed to cell proliferation were found to be upregulated by If the expression of Hdc gene has a physiological sig- fibrates at the time when Hdc was induced (Tamura et al., nificance, it should be translated to protein and this was 2006; Mizukawa et al., 2020). confirmed by western blotting. HDC protein has a size of HDC is practically absent in the adult rat liver, but 74 kDa in full length and is known to be processed after highly expressed in the liver of fetuses (Taguchi et al., translation (Ichikawa et al., 2010; Tanaka et al., 1998; 1984) and considered to be involved in cell prolifera- Furuta et al., 2007). The 74 kDa precursor is labile and tion during development or regeneration like after partial easily destroyed by ubiquitin-proteasome, while its C-ter- hepatectomy (Ishikawa et al., 1970). Chang et al. (2010) minal fragment is cleaved off by caspase 9 to make the investigated expression changes in the gene related to his- 54 kDa active form on the cytoplasmic reticulum mem- tidine catabolism in the proliferating liver using a partial brane. This was known from an analysis by the transfec- hepatectomy model of rat. They showed that expression tion of fragments into cell lines, but not in the physiolog- of Hdc was induced in hepatocytes, vascular endotheli- ical condition. In the present study, the interpretation of al cells and bile duct epithelia, and the expression of Hal the results was difficult because HDC protein was quite was increased in Kupffer cells under the increased hepat- unstable especially under the solubilized condition such ic proliferation by hepatectomy. They suggested that his- that unreproducible bands appeared in the western blot- tamine produced by the former pathway stimulates sinu- ting using two types of commercially available antibod- soidal endothelial cells proliferation,and urocanic acid ies. According to the manufacturers’ data sheets, anti- produced by the latter acts on Kupffer cells itself and den- body from Abcam (Ab-A) recognizes C-terminal and dritic cells to generate immune suppression by autocrine that from Atlas (Ab-B) recognizes N-terminal portion of and paracrine modes. In the present study, the upregula- HDC. Repeated experiments using freshly prepared sam- tion of Hdc was quite obvious whereas the expression of ples to get relatively reproducible results indicated that a Hal was rather decreased. This could be due to the differ- 30 kDa fragment was detected by Ab-A in the cytosol and ence that the data from the database are the measurements 74 (faint) and 65 kDa bands were detected by Ab-B in of whole liver, while the measurements by Chang et al. the membrane fraction of gastric mucosa. As the appar- (2010) were done on the separated cell types. In fact, ent difference on the SDS-PAGE was within a reasonable expression of Hal in the hepatocyte fraction was marked- range, it could be suggested that the processing of HDC ly decreased 3 days after the hepatectomy in their data. in the rat gastric mucosa occurs according to the previ- Considering the reports of hepatectomy described ous reports (Ichikawa et al., 2010; Tanaka et al., 1998; above, it could be postulated that compensatory cell pro- Furuta et al., 2007), i.e., the full length form and its acti- liferation due to primary tissue damage by fibrates leads vated form are on the membrane leaving the C-terminal to the activation of Hdc expression. However, upregula- fragment in the cytosol. On the other hand, the pattern tion of Hdc expression was almost limited to the drugs was different in the brain samples. Ab-A only recognized having PPARα stimulation, whereas there were drugs a 74 kDa band in the membrane fraction, whereas Ab-B causing cell damage followed by hepatic hypertrophy recognized 75 (faint) and 45 kDa bands in the membrane without inducing Hdc expression. For example, TAA fraction and a 110 kDa band in the cytosol of rat brain. or CMA, which caused severe necrosis (Uehara et al., It appears that the processing of HDC is different in the 2008a, 2008b) and subsequent increase in relative liver brain and it is difficult to interpret the results by Ab-B, weight per body weight (Table 2) showed no upregulation and we did not elucidate this issue further in the present of Hdc. It is thus suggested that the expression of Hdc study. In any event, HDC induced in the liver by fibrates associated with cell proliferation is not the result of tis- appears to be processed as in the gastric mucosa except sue damage, but is directly related to PPARα stimulation. for the fact that a small amount of C-terminal fragment is Mitogens are reported to induce HDC in the liver (Endo, remained in the membrane fraction. 1983) although this is only in the study of mice. Taken It is known that hepatomegaly caused by PPARα ago- together, it is concluded that Hdc induction appeared to nists is due to the increase in the cell size associated with be restricted to the direct stimulation of cell proliferation enzyme induction as well as that in cell proliferation by mitogens, hepatectomy, or PPARα stimulation. The

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Fig. 11. Effects of FFB and WY on the expression of Hdc in mouse liver and on the relative liver weight per body weight (%). A: Mice were received 100 mg/kg FFB or vehicle for 3 and 7 days (N=4 each), and the expression of Hdc was quantified by real-time PCR, and normalized by β-actin. B: Liver weight per body weight (%) of the mice in A. C: Mice were adminis- tered 100 mg/kg WY or vehicle for 3 days (N=3 each), and the expression of Hdc was quantified by real-time PCR, and normalized by β-actin. D: Liver weight per body weight (%) of the mice in C. The data are expressed as mean + SEM. * Significantly different from control by Student’s t-test at p < 0.05. expression of Hal did not appear to be closely related to 2010), most of which are removed from the culture. How- hepatic hypertrophy in general, but it seemed to be self- ever, in the case of Hdc, it was found to be expressed in regulation to compensate for the reduction of histidine in the parenchymal cells by immunostaining. Therefore, it the hepatocyte under PPARα stimulation. is necessary to consider the involvement of other factors, In order to investigate the direct relation between the such as autonomic nerves, endocrine/paracrine system, or Hdc expression and cell proliferation induced by PPARα something from food, to explain this large difference. agonists, primary cultured hepatocytes should be a useful Another possibility is that the gene expression is stim- tool. However, the effects of PPARα agonists could not be ulated by separation and/or cultivation of the hepatocytes reproduced in vitro at all. As previously reported (Tamura such that the stimulatory effects by drugs are masked. This et al., 2006; Mizukawa et al., 2020), the genes extensive- type of phenomenon was observed in the case of cell pro- ly upregulated by fibrates contained a large number of the liferation-related genes in the previous study (Mizukawa genes that did not show any expression changes in vitro. et al., 2020). In the present study, Slc27a2 expression was This cannot be explained by fast metabolic elimination of stimulated by fibrates as early as 3 hr after administration fibrates in vitro, since metabolic enzymes in vitro are rap- in vivo (Fig. 4), whereas their effects were only notice- idly decreased (Berry et al., 1997) and found to be less able after 24 hr in vitro (Fig. 5). Looking at the control stimulated by fibrates compared with that in vivo (Tamura values, they decreased with time of culture such that the et al., 2006). Moreover, if the difference is due to phar- stimulatory effects emerged at 24 hr. However, this was macokinetics, the difference should be even for all of the not the case for Hdc, since its expression was kept very genes. low throughout the culture time. In general, in vivo specific responses are attributed After the cultured cell system was found to be inap- to the change in the non-parenchymal cells (Qu et al., propriate, we were obliged to employ another strategy.

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PPARα-induced HDC gene expression in the rat and mouse liver

There are various gene-targeting mice available including in the hepatic hypertrophy, considering the known role Hdc-knockout (Ichikawa et al., 2010; Ohtsu et al., 2001), of histamine in the proliferation of various cell types in histamine receptor-knockout (Ichikawa et al., 2010; rodents (Ichikawa et al., 2010; Medina and Rivera, 2010). Masaki et al., 2005), or PPARα-knockout mice (Lee et At present, the most feasible interpretation would be that al., 1995), and the combination of these should be a pow- the hypertrophy caused by PPARα agonists is mainly due erful tool. Before using these mice, we administered FFB to their well-known enzyme-inducing effect (Maronpot or WY to wild-type mice, but unexpectedly, no induc- et al., 2010), and the physiological role of Hdc induction tion of Hdc occurred. As HDC activity is reported to be on cell proliferation is possible but still unknown. Fur- induced by mitogens in mice (Endo, 1983), proliferation- ther study is necessary for comprehensive understanding related induction of Hdc should also exist in mice. Fur- of the pharmacology and toxicology of PPARα agonists, thermore, both the rat and the mouse, as rodents, have and we believe that the present results contribute to the been commonly used in pharmacological as well as tox- research providing a new clue. icological research (Desvergne and Wahli, 1999). This inconceivable result cannot be explained by the difference ACKNOWLEDGMENTS in the metabolic enzymes for fibrates because the change of the drug concentration is expected to affect all of the We thank Prof. Atsushi Ono (Okayama University) for genes evenly. fruitful discussion. We thank Mr. John H. Jennings for It is well known that the expression of a certain gene editing manuscript. affects that of its neighboring gene, and in most cases, the nearest pair genes interact with each other (Sémon and Conflict of interest---- The authors declare that there is Duret, 2006). Among them, “PROMPT”, where the pro- no conflict of interest. moter regions of two neighboring genes exist in a head- to-head position in the complementary strands and both REFERENCES genes are transcribed at the same time, has been exten- sively studied (Lloret-Llinares et al., 2016; Mizukawa Aggerbeck, M., Ferry, N., Zafrani, E.-S., Billon, M.-C., Barouki, R. et al., 2020). In the case of rat Hdc, the nearest one is and Hanoune, J. (1983): Adrenergic regulation of glycogenolysis in rat liver after cholestasis. Modulation of the balance between PPARα-inducible Slc27a2 (Rakhshandehroo et al., 2010), alpha 1 and beta 2 receptors. J. Clin. 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