effect of structural modifications on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced...

Research Article

Received: 30 June 2010, Revised: 1 November 2010, Accepted: 17 November 2010 Published online in Wiley Online Library: 21 February 2011

(wileyonlinelibrary.com) DOI 10.1002/jat.1639

108

Effect of structural modifications on3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione‐induced hepatotoxicityin Fischer 344 ratsNiti N. Patel, Christine M. Crincoli, Douglas M. Frederick,Ruy Tchao and Peter J. Harvison*

ABSTRACT: Glitazones, used for type II diabetes, have been associated with liver damage in humans. A structural featureknown as a 2,4‐thiazolidinedione (TZD) ring may contribute to this toxicity. TZD rings are of interest since continued humanexposure via the glitazones and various prototype drugs is possible. Previously, we found that 3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione (DCPT) was hepatotoxic in rats. To evaluate the importance of structure on DCPT toxicity, we thereforestudied two series of analogs. The TZD ring was replaced with: a mercaptoacetic acid group {[[[(3,5‐dichlorophenyl)amino]carbonyl]thio]acetic acid, DCTA}; a methylated TZD ring [3‐(3,5‐dichlorophenyl)‐5‐methyl‐2,4‐thiazolidinedione, DPMT]; andisomeric thiazolidinone rings [3‐(3,5‐dichlorophenyl)‐2‐ and 3‐(3,5‐dichlorophenyl)‐4‐thiazolidinone, 2‐DCTD and 4‐DCTD,respectively]. The following phenyl ring‐modified analogs were also tested: 3‐phenyl‐, 3‐(4‐chlorophenyl)‐, 3‐(3,5‐dimethylphenyl)‐ and 3‐[3,5‐bis(trifluoromethyl)phenyl]‐2,4‐thiazolidinedione (PTZD, CPTD, DMPT and DFMPT, respectively).Toxicity was assessed in male Fischer 344 rats 24 h after administration of the compounds. In the TZD series only DPMTproduced liver damage, as evidenced by elevated serum alanine aminotransferase (ALT) activities at 0.6 and 1.0 mmol kg−1

(298.6 ± 176.1 and 327.3 ± 102.9 Sigma‐Frankel units ml−1, respectively) vs corn oil controls (36.0 ± 11.3) and morphologicalchanges in liver sections. Among the phenyl analogs, hepatotoxicity was observed in rats administered PTZD, CPTD andDMPT; with ALT values of 1196.2 ± 133.6, 1622.5 ± 218.5 and 2071.9 ± 217.8, respectively (1.0 mmol kg−1 doses).Morphological examination revealed severe hepatic necrosis in these animals. Our results suggest that hepatotoxicity ofthese compounds is critically dependent on the presence of a TZD ring and also the phenyl substituents.Copyright © 2011John Wiley & Sons, Ltd.

Keywords: 3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione; hepatotoxicity; rat; structure–activity relationship; thiazolidinedione

*Correspondence to: P. J. Harvison, Department of Pharmaceutical Sciences,University of the Sciences in Philadelphia, Philadelphia, PA 19104, USA.E‐mail: [email protected]

Department of Pharmaceutical Sciences, University of the Sciences inPhiladelphia, Philadelphia, PA 19104, USA

INTRODUCTION

As part of an investigation into the potential toxicity of cyclicimide containing compounds, we previously found that 3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione (DCPT, Fig. 1) producedliver damage in male rats (Kennedy et al., 2003). We are interestedin this compound because it contains a 2,4‐thiazolidinedione (TZD)ring. This structural feature is also present in the ‘glitazone’ insulin‐sensitizing agents that were originally developed for the treatmentof type II diabetes. Troglitazone (Fig. 1), the first member of thisclass to be marketed, was associated with liver damage in diabeticpatients and was removed from the market (Gitlin et al., 1998;Kohlroser et al., 2000; Graham et al., 2003). Rosiglitazone andpioglitazone are still used clinically, although there have beenseveral reports of mild hepatic injury with both of these drugs(Gouda et al., 2001; Maeda, 2001; Marcy et al., 2004; El‐Naggar et al.,2008). As a class, the glitazones are not recommended for use inpatients with existing liver disease (Scheen, 2001). TZD derivativesare also being investigated as potential aldose reductase inhibi‐tors for the treatment of diabetic complications (Bruno et al., 2002;Rakowitz et al., 2006), analgesic and anti‐inflammatory agents(Ali et al., 2007) and androgen antagonists (Yang et al., 2008).

J. Appl. Toxicol. 2012; 32: 108–117 Copyright © 2011 John

Upon further investigation, we found that DCPT‐inducedhepatotoxicity in rats was dependent on time, dose and gender(Patel et al., 2008). Liver damage, seen as morphological changesand elevations in serum alanine aminotransferase (ALT) levels, wasapparent within 3 h of dosing and was fully established at 24 h inmale rats. In comparison, female rats were less susceptible tohepatotoxicity thanmales (Patel et al., 2008), which couldbedue togender‐dependent differences in metabolism (Mugford andKedderis, 1998; Czerniak, 2001). In separate studies, we evaluatedthe potential role of cytochromes P450 (CYPs) in DCPT‐inducedliver damage (Crincoli et al., 2008). Both 1‐aminobenzotriazole(non‐specific CYP inhibitor) and troleandomycin (CYP3A inhibitor)attenuated DCPT toxicity. In contrast, the CYP3A inducerdexamethasone potentiated hepatic injury. Thus, it seems that a

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Figure 1. Structures of thiazolidinedione ring‐containing compounds. Calculated log P values for DCPT and its analogs are shown in parentheses.

Effect of structual modifications on DCPT hepatotoxicity in rats

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CYP3A‐derived DCPT metabolite is responsible for the hepatotox-icity of this compound in male rats (Crincoli et al., 2008).

As noted above, TZD rings are found in rosiglitazone,pioglitazone and several prototype therapeutic agents. Thus,there is potential for continued human exposure to compoundsthat contain this structural feature. As a consequence, it isimportant to further explore the potential role of TZD rings inchemically induced hepatotoxicity. Towards this goal, wedecided to conduct a structure–activity relationship (SAR)study. We therefore synthesized, characterized and evaluatedthe potential toxicity of two different series of DCPT analogs: (1)compounds that contained modifications to the TZD ring, butretained the 3,5‐dichlorophenyl ring; and (2) compounds thatretained the TZD ring, but contained different substituents inthe phenyl ring. For the first series of compounds (Fig. 1), theTZD ring was replaced with: its potential hydrolysis product, amercaptoacetic acid group {[[[(3,5‐dichlorophenyl)amino]carbonyl]thio]acetic acid, DCTA}; an alkyl substituted TZD ring[3‐(3,5‐dichlorophenyl)‐5‐methyl‐2,4‐thiazolidinedione, DPMT];and isomeric thiazolidinone rings [3‐(3,5‐dichlorophenyl)‐2‐thiazolidinone and 3‐(3,5‐dichlorophenyl)‐4‐thiazolidinone,2‐DCTD and 4‐DCTD, respectively]. In the second set of analogs,phenyl substituents were investigated that could exert electron‐releasing or electron‐withdrawing effects on the TZD ring, whichcould potentially alter its metabolism and hence toxicity.Therefore, we also evaluated the following phenyl‐substitutedDCPT analogs (Fig. 1): 3‐phenyl‐2,4‐thiazolidinedione (PTZD),3‐(4‐chlorophenyl)‐2,4‐thiazolidinedione (CPTD), 3‐(3,


5‐dimethylphenyl)‐2,4‐thiazolidinedione (DMPT) and 3‐[3,5‐bis(trifluoromethyl)phenyl]‐2,4‐thiazolidinedione (DFMPT). Basedon our results, it can be concluded that an intact TZD ring is animportant structural feature for hepatotoxicity and that phenylring substituents can alter toxicity of this series of compounds.

MATERIALS AND METHODS

Chemicals/Reagents

The starting materials 3,5‐dichlorophenyl isocyanate, 3,5‐dichlorophenyl thiourea and 3,5‐dichloroaniline wereobtained from Alfa Aesar (Ward Hill, MA, USA). Phenylisocyanate, 3,5‐dimethylphenyl isocyanate, 4‐chlorophenylisocyanate, 3,5‐bis(trifluoromethyl)phenyl isocyanate, ethyl‐2‐mercaptoacetate, ethyl‐2‐mercaptopropionate, 1,2‐dibromoethane and mercaptoacetic acid were purchasedfrom Sigma‐Aldrich Chemical Co. (Milwaukee, WI, USA). Bio‐Rad protein assay kit was obtained from Bio‐Rad Laboratories(Hercules, CA, USA). 2,4‐Dinitrophenylhydrazine, sodiumpyruvate, DL‐alanine, α‐keto‐glutaric acid, alanine aminotrans-ferase assay kit (ALT, no. 505‐P), blood urea nitrogen assay kit(BUN, no. 640‐5) and urine/blood glucose assay kit (no. 510‐DA) were all products of Sigma Chemical Co. (St Louis, MO,USA). These assay kits were later discontinued by themanufacturer. In some experiments, the ALT assay wastherefore done using a procedure similar to that describedin the Sigma kit. The required solutions were alanine (0.2 M)/

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N. N. Patel et al.

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α‐ketoglutarate (1.8 mM) in 100 mM sodium phosphate buffer,pH 7.4; 2,4‐dinitrophenylhydrazine (20 mg dl−1) in 1 M HCl;and sodium pyruvate (1.5 mM) in 100 mM sodium phosphatebuffer, pH 7.4. A standard curve (0, 23, 50, 83,125 Sigma‐Frankel units ml−1) was generated by combining the sodiumpyruvate (0, 0.5, 0.1, 0.15, 0.2 ml) and alanine/α‐ketoglutaratesolutions (0.5, 0.45, 0.4, 0.35, 0.3 ml) with water (0.1 ml).Serum samples and standards (0.1 ml) were incubated withthe alanine/α‐ketoglutarate solution (0.5 ml) for 30 min at37 °C before addition of 2,4‐dinitrophenylhydrazine reagent(0.5 ml). After standing at room temperature for 20 min, 0.4 M

NaOH (5 ml) was added and absorbance was measured at505 nm. Samples outside the range of the standard curvewere diluted 1:10 or 1:20 with water before being re‐assayed.The BUN assay (no. B7551‐120) and urine/blood glucose assay(no. G7518‐150) were replaced with the indicated kitsobtained from Pointe Scientific (Detroit, MI, USA). Experi-ments were conducted to ensure that the assay results fromthe different kits were comparable.

Animals

Male Fischer 344 rats (ca 200 g) were purchased from CharlesRiver Laboratories (Wilmington, MA, USA). The rats wereindividually housed in standard stainless steel hanging cagesunder a 12 h light/dark cycle at ca 22 °C and 45–50% relativehumidity. Food (laboratory rodent diet no. 5001, PMI Foods Inc.,St Louis, MO, USA) and water were freely available unlessotherwise noted. The animals were held in the Vivarium for aminimum of one week before use in any experiments. Allexperiments involving the rats were approved by the Institu-tional Animal Care and Use Committee of the University of theSciences in Philadelphia.

Syntheses

Melting points were determined with a Thomas–Hoovercapillary melting point apparatus and are uncorrected. 1 HNMR and 13 C NMR spectra were recorded at 400 and 100 MHz,respectively, on a Bruker Avance 400 nuclear magneticresonance (NMR) spectrometer. Chemical shifts (δ) are reportedin parts per million (ppm) relative to tetramethylsilane as theinternal standard. The abbreviations used in reporting thesplitting patterns are as follows: s (singlet), d (doublet), t (triplet),q (quartet) and m (multiplet). Elemental analyses wereperformed by Galbraith Laboratories Inc. (Knoxville, TN, USA).

Preparation of TZD Ring‐modified Compounds

The methyl TZD ring compound, 3‐(3,5‐dichlorophenyl)‐5‐methyl‐2,4‐thiazolidinedione (DPMT), was synthesized by amodification of the method of Fujinami et al. (1971) andKennedy et al. (2003). In step 1, ethyl 2‐[[[(3,5‐dichlorophenyl)amino]carbonyl]thio]propionate was prepared by refluxing 3,5‐dichlorophenyl isocyanate (10.0 g, 0.053 mol) with ethyl2‐mercaptopropionate (6.96 g, 0.052 mol) and pyridine(0.114 ml) in toluene (32 ml) for 22 h. The reaction mixturewas cooled in a freezer and the intermediate product wascollected and dried in a vacuum oven. For step 2, ethyl 2‐[[[(3,5‐dichlorophenyl)amino]carbonyl]thio]propionate (5 g, 0.016 mol)and triethylamine (0.25 g, 0.0024 mol) were refluxed in toluenefor 24 h. After cooling the reaction mixture in a freezer, the

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precipitate was filtered and discarded. The filtrate wasconcentrated on a rotary evaporator to afford the crudeproduct. DPMT was purified by recrystallization using a mixedsolvent system of ethanol–water; m.p. 157.5–158.5 °C. 1 H NMR(DMSO‐d6): δ 7.78 (d, 2H, Ar–H2,6), 7.57 (t, 1H, Ar–H4), 4.63 (q, 1H,CH) and 1.67 (d, 3H, CH3).

13 C NMR (DMSO‐d6): δ 175.56(C=O), 171.79 (C=O), 136.77 (Ar–C3,5), 134.80 (Ar–C1), 129.72(Ar–C4), 128.14 (Ar–C2,6), 45.02 (CH) and 18.82 (CH3). Anal.calcd for C10H7Cl2NO2S (276.13): C, 43.50; H, 2.56; N, 5.07; O,11.59; S, 11.61; Cl, 25.68. Found: C, 43.54; H, 2.51; N, 5.04;S, 11.71; Cl, 26.41.

A modification of the method of Fujinami et al. (1968) wasfollowed for the preparation of DCTA. Briefly, 3,5‐dichlorophenylisocyanate (9.4 g, 0.05 mol), pyridine (0.2 ml) and benzoylperoxide (0.005 g) were dissolved in 25 ml tetrahydrofuran (THF).Mercaptoacetic acid (4.6 g, 0.05 mol) in THF (25 ml) was thenadded dropwise and the reaction mixture was refluxed at 55 °Cfor 1 h. After cooling in a freezer, the precipitate was collected anddiscarded. The filtrate was concentrated on a rotary evaporatorto yield the crude product. DCTA was recrystallized usingabsolute ethanol; m.p. 139–140 °C [lit. m.p. 122–124.5 °C (Fujinamiet al., 1968)]. 1 H NMR (DMSO‐d6): δ 12.08 (s, 1H, COOH), 10.85(s, 1H, NH), 7.53 (d, 2H, Ar–H2,6), 7.29 (t, 1H, Ar–H4) and 3.73 (s, 2H,CH2).

13 C NMR (DMSO‐d6): δ 170.82 (C=O), 165.75 (C=O), 143.69(Ar–C3,5), 134.95 (Ar–C1), 123.58 (Ar–C4), 117.81 (Ar–C2,6) and32.70 (CH2). Anal. calcd for C9H6Cl2NO3S (279.11): C, 38.71; H, 2.17;N, 5.02; O, 17.20; S, 11.49; Cl, 25.40. Found: C, 38.76; H, 2.55; N, 5.00;S, 11.43; Cl, 25.22.

The two‐step procedure of Brouwer and Blem (1987) was usedfor the synthesis of 4‐DCTD. First, 3,5‐dichloroaniline (20.12 g,0.125 mol) and mercaptoacetic acid (11.5 g, 0.125 mol) werecombined in 95% ethanol with stirring on ice. Formalin solution(10.65 g, 37% by weight) was then added dropwise which led tothe formation of a white precipitate. After stirring for 24 h, water(15 ml) was added to the reaction mixture and stirring wascontinued for several hours. The precipitated compound, 2‐[[(3,5‐dichlorophenyl)amino]methyl]thioacetic acid, was filtered and airdried. This intermediate (30 g, 0.112 mol) was then refluxed inxylene (217 ml) for 6 h with azeotropic removal of water (Dean–Stark trap) to yield 4‐DCTD. The crude 4‐DCTD was recrystallizedusing absolute ethanol; m.p. 103.5–105.5 °C [lit. m.p. 87–92 °C(Brouwer and Blem, 1987)]. 1 H NMR (DMSO‐d6): δ 7.68 (d, 2H,Ar–H2,6), 7.48 (t, 1H, Ar–H4), 4.93 (s, 2H, CH2) and 3.73 (s, 2H, CH2).13 C NMR (DMSO‐d6): δ 172.22 (C=O), 142.20 (Ar–C3,5), 134.90(Ar–C1), 125.57 (Ar–C4), 120.93 (Ar–C2,6), 49.73 (CH2) and 33.19(CH2). Anal. calcd for C9H7Cl2NOS (248.12): C, 43.57; H, 2.84; N,5.64; O, 6.45; S, 12.92; Cl, 28.58. Found: C, 43.76; H, 3.06; N, 5.68; S,12.87; Cl, 29.60.

Synthesis of 2‐DCTD proceeded in two steps. The intermediate2‐methylimino‐3‐(3,5‐dichlorophenyl)thiazolidine was first syn-thesized using a variation on the method of Toldy et al. (1972).Briefly, 3,5‐dichlorophenyl thiourea (10 g, 0.045 mol) and 1,2‐dibromoethane (19.27 g, 0.013 mol) were refluxed in isoamylalcohol (25 ml) for 4 h. The resulting intermediate, 2‐methylimino‐3‐(3,5‐dichlorophenyl)thiazolidine was hydrolyzed to 2‐DCTD viathe diazonium salt, using a modification of the method of Tashiroand Nakamura (1985). Thus, 2‐methylimino‐3‐(3,5‐dichlorophenyl)thiazolidine (4.5 g, 0.018 mol) was dissolved in water (20 ml), thesolution was acidified (pH 2.5–3.5) with HCl and xylene (20 ml)was added. The reaction mixture was heated to 60 °C for 30 minand then aqueous sodium nitrite (5 ml, 5% solution) was added.The temperature was raised to 90 °C and heating was continued

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until the color of the reaction mixture became pale yellow. Thecrude product (2‐DCTD) was extracted with xylene and thesolvent was removed on a rotary evaporator. 2‐DCTD waspurified by column chromatography on silica gel (60–200 mesh)using benzene and then benzene–hexane (9:1) as eluents; m.p.105–106 °C. 1 H NMR (DMSO‐d6): δ 7.56 (d, 2H, Ar–H2,6), 7.42(t, 1H, Ar–H4), 4.21 (t, 2H, CH2) and 3.46 (t, 2H, CH2).

13 C NMR(DMSO‐d6): δ 172.17 (C=O), 141.89 (Ar–C3,5), 134.92 (Ar–C1),124.91 (Ar–C4), 120.54 (Ar–C2,6), 50.88 (CH2) and 25.85 (CH2). Anal.calcd for C9H7Cl2NOS (248.12): C, 43.57; H, 2.84; N, 5.64; O, 6.45;S, 12.92; Cl, 28.58. Found: C, 44.00; H, 3.13; N, 5.68; S, 13.18;Cl, 29.31.

Preparation of Phenyl Ring‐modified Compounds

All analogs with modifications in the phenyl ring weresynthesized similarly to DPMT. In brief, phenyl isocyanate orappropriately substituted derivatives were condensed with ethyl2‐mercaptoacetate, followed by base‐catalyzed cyclization tothe TZD ring‐containing compounds.

Recrystallization of the unsubstituted analog, PTZD using 95%ethanol yielded pure product; m.p. 142.5–143.5 °C. 1 H NMR(DMSO‐d6): δ 7.52‐7.30 (m, 5H, Ar–H), 4.31 (s, 2H, CH2).

13 C NMR(DMSO‐d6): δ 172.84 (C=O), 172.28 (C=O), 134.18 (Ar–C3,5), 129.98(Ar–C1), 129.78 (Ar–C4), 128.75 (Ar–C2,6) and 34.28 (CH2). Anal.calcd for C9H7NO2S (193.22): C, 55.95; H, 3.65; N, 7.25; O, 16.56;S, 16.59. Found: C, 55.96; H, 3.55; N, 7.22; S, 17.01.

Purification of DMPT was accomplished via recrystallizationusing 95% ethanol; m.p. 183–184 °C. 1 H NMR (DMSO‐d6): δ 7.10(s, 1H, Ar–H4), 6.90 (s, 2H, Ar–H2,6), 4.29 (s, 2H, CH2) and 2.30(s, 6 H, Ar–CH3).

13 C NMR (DMSO‐d6): δ 172.73 (C=O), 172.26(C=O), 139.30 (Ar–C3,5), 134.12 (Ar–C1), 131.06 (Ar–C4), 126.80(Ar–C2,6), 34.28 (CH2) and 24.03 (Ar–CH3). Anal. calcd forC11H11NO2S (221.27): C, 59.71; H, 5.01; N, 6.33; O, 14.46; S,14.40. Found: C, 59.78; H, 5.08; N, 6.22; S, 14.38.

The para‐subsituted compound, CPTD, was recrystallized using amixed solvent system of absolute ethanol‐water; m.p. 143–144 °C.1 H NMR (DMSO‐d6): δ 7.59 (m, 2H, Ar–H3,5), 7.37 (m, 2H, Ar–H2,6)and 4.29 (s, 2H, CH2).

13 C NMR (DMSO‐d6): δ 172.65 (C=O),172.03 (C=O), 134.39 (Ar–C3,5), 132.08 (Ar–C1), 130.54 (Ar–C4),130.00 (Ar–C2,6) and 35.22 (CH2). Anal. calcd for C9H6ClNO2S(227.66): C, 47.48; H, 2.66; N, 6.15; O, 14.06; S, 14.08; Cl, 15.57. Found:C, 47.31; H, 2.70; N, 5.91; S, 14.16; Cl, 15.66.

Purification of DFMPT was accomplished via recrystallizationfrom hexane; m.p. 115–116 °C. 1 H NMR (DMSO‐d6): δ 8.28 (s, 1H,Ar–H4), 8.18 (s, 2H, Ar–H2,6) and 4.32 (s, 2H, CH2).

13 C NMR(DMSO‐d6): δ 172.56, 171.80, 136.09, 132.11 (q), 130.21, 127.75,125.04, 123.73 (d), 122.33, 119.62 and 35.45. Anal. calcd forC11H5F6NO2S (329.21): C, 40.13; H, 1.53; N, 4.25; O, 9.72; S, 9.74; F,34.62. Found: C, 40.37; H, 1.57; N, 4.27; S, 9.72; F, 34.43.

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In Vivo Toxicity

The experimental method is similar to our previously published24 h procedure (Patel et al., 2008). Rats were randomly dividedinto treatment groups (N=3–4 rats/group) and transferred toindividual stainless steel metabolism cages (Allentown CagingEquipment Co., Allentown, NJ, USA) to allow for the collection ofurine. The animals were kept in the metabolism cages for aminimum of 2 days before dosing.

On the first day of the experiment (control day, before anytreatments), urine was collected for 6 h during which food and


water were removed from the cages to avoid contamination ordilution of the urine sample. All 6 h urine samples were stored at−78 °C for quantitative determination of protein levels. After the6 h period, food (ca 40 g) and water (150 ml) were returned intothe cages. Food and water intake, and the amount of urineexcreted over the next 18 h were also measured.On day 2, each animal received a single dose of a DCPT

analog (0.2, 0.4, 0.6 or 1.0 mmol kg−1, i.p. in corn oil) or corn oilonly (4 ml kg−1). Dosages >1.0 mmol kg−1 are generallyimpractical due to solubility problems. Rats treated with DCTAat 0.6 mmol kg−1 appeared to be moribund (sluggish move-ments and low body temperatures) 24 h after dosing; therefore,this compound was only evaluated at three doses (0.2, 0.4 and0.6 mmol kg−1, i.p. in corn oil). All animals were returned to themetabolism cages immediately after treatment and urine wascollected as described above. Food and water intake, and theamount of urine excreted over the next 18 h were alsomeasured. Twenty‐four hours after dosing, the rats wereanesthetized using isoflurane. While under anesthesia, a bloodsample was obtained by cardiac puncture and then the animalswere euthanized by cervical dislocation. The blood sampleswere allowed to clot for 15 min at room temperature andcentrifuged at 12 000 rpm for 20 min. Analysis for alanineaminotransferase (ALT) levels was conducted using fresh serumand the serum samples were then stored at −78 °C forsubsequent analysis of blood urea nitrogen (BUN) levels. Thekidneys and livers were removed, weighed, and the right kidneyand a section of liver were fixed in formalin for histologicalanalysis. Tissue sections were prepared and stained withhematoxylin and eosin (H&E) by American Histolabs Inc.(Gaithersburg, MD, USA).

Computational Analyses

As an estimate of relative lipophilicities, log P values werecalculated using the ChemDraw Ultra 8.0 software package(CambridgeSoft, Cambridge, MA, USA).

Statistics

Results are expressed as means ± SE (N= 3 or 4). The data wereanalyzed by a one‐way ANOVA followed by a Student–Newman–Keuls post hoc test. When the normality or equalvariance tests failed, the data were analyzed by a one‐wayANOVA on ranks. Differences in the means were consideredsignificant when P< 0.05.

RESULTS

TZD Ring‐modified Compounds

Serum ALT levels (Fig. 2) were measured in all rats as an index ofliver damage. Compared with the two lower doses and corn oilcontrols, rats that received DPMT at 0.6 and 1.0 mmol kg−1

exhibited a significant elevation in ALT concentrations. Admin-istration of DCTA at 0.6 mmol kg−1, which resulted in reducedbody temperatures and sluggish movements in the animals,produced a modest increase (about 2‐fold) in ALT levels, but at0.2 and 0.4 mmol kg−1 no changes were noted. 4‐DCTD‐ and 2‐DCTD‐treated animals did not exhibit any significant changes inserum ALT levels at any of the doses evaluated. No significantchanges were observed in liver weights with any of the


1

Figure 2. Effect of thiazolidinedione ring modifications on serum alanine aminotransferase (ALT) levels in rats. Compounds were administered i.p. incorn oil. Controls received corn oil only (4 ml kg−1). Values are means ± SE (N=3–4). An asterisk indicates that the value is significantly different(P<0.05) from the corn oil control. Different letters within a treatment group indicate that the doses are significantly different (P<0.05).

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treatments when compared across doses for a compound or tothe corn oil controls (Table 1).

Representative photomicrographs of liver sections from theanimals treated with corn oil or a TZD‐ring modified DCPT

Table 1. Effect of thiazolidinedione ring modifications on bloodkidney weights in rats

Treatment Dose(mg kg−1)

Blood urea nitrogen(mg dl−1)

Twenty‐four‐hoururine volume

(ml)

Corn Oil — 20.2 ± 2.0 3.7 ± 0.5

DPMT 0.2 19.6 ± 0.8a 6.5 ± 2.1a

0.4 24.6 ± 1.3a 8.1 ± 1.7a

0.6 19.8 ± 2.8a 11.6 ± 1.0*,a

1.0 20.0 ± 2.2a 10.8 ± 1.3*,a

DCTA 0.2 21.0 ± 1.0b 12.5 ± 3.8b

0.4 62.2 ± 11.2*,c 13.1 ± 2.4b

0.6 67.8 ± 17.9*,c 9.5 ± 5.0b

4‐DCTD 0.2 26.8 ± 1.5d 12.3 ± 1.7*,c

0.4 25.6 ± 1.9d 11.3 ± 2.2*,c

0.6 33.8 ± 2.4*,e 9.1 ± 1.6*,c

1.0 42.5 ± 1.1*,f 12.3 ± 1.0*,c

2‐DCTD 0.2 23.0 ± 1.2g 6.0 ± 0.8d

0.4 22.3 ± 1.1g 4.3 ± 0.3d

0.6 23.9 ± 0.9g 5.0 ± 0.7d

1.0 24.4 ± 0.7g 4.4 ± 1.3d

Compounds were administered i.p. in corn oil. Controls receivedasterisk indicates that the value is significantly different (P<0.05)group indicate that the doses are significantly different (P< 0.05).


analog (highest dose for each compound) are shown in Fig. 3.The liver from a corn oil‐treated animal appeared normal(Fig. 3A). Hepatocytes were healthy in appearance and werearranged in a cord‐like fashion radiating out of the central veins.

urea nitrogen, urine volume and protein content, or liver and

Six‐hour urineprotein content

(mg)

Liver weight(g per 100% b.w.)

Kidney weight(g per 100% b.w.)

1.5 ± 0.4 3.49 ± 0.12 0.37 ± 0.01

2.1 ± 0.7a 3.67 ± 0.31a 0.37 ± 0.01a

1.6 ± 0.1a 3.81 ± 0.22a 0.38 ± 0.01a

2.6 ± 0.8a 4.03 ± 0.27a 0.39 ± 0.01a

2.5 ± 0.3a 4.10 ± 0.07a 0.41 ± 0.01a

2.9 ± 0.9b 3.58 ± 0.21b 0.40 ± 0.01b

2.5 ± 0.4b 3.62 ± 0.05b 0.43 ± 0.01*,b,c

2.5 ± 1.1b 3.46 ± 0.17b 0.45 ± 0.01*,c

1.8 ± 0.3c 3.74 ± 0.23c 0.40 ± 0.01d

1.6 ± 0.2c 3.96 ± 0.18c 0.37 ± 0.01d

1.9 ± 0.4c 3.46 ± 0.09c 0.41 ± 0.01d

2.8 ± 0.3c 3.81 ± 0.15c 0.42 ± 0.01d

1.6 ± 0.4d 3.96 ± 0.10d 0.39 ± 0.01e

1.8 ± 0.1d 3.71 ± 0.11d 0.38 ± 0.01e

2.2 ± 0.1d 3.74 ± 0.11d 0.40 ± 0.01e

2.0 ± 0.1d 3.85 ± 0.15d 0.37 ± 0.01e

corn oil only (4 ml kg−1). Values are means ± SE (N=3–4). Anfrom the corn oil control. Different letters within a treatment


Figure 3. Effect of thiazolidinedione ring modifications on rat hepaticmorphology. Representative photomicrographs (A, corn oil; B, DPMT; C,DCTA; D, 4‐DCTD; E, 2‐DCTD) are shown for the highest dose administeredfor each compound (1.0 mmol kg−1 for DPMT, 2‐DCTD and 4‐DCTD;0.6 mmol kg−1 for DCTA; i.p. in corn oil). Controls received corn oil only(4 ml kg−1). Vacuolated cells and swollen hepatocytes are indicated bywhite arrows and black arrows, respectively. Magnification is 400×.


11

Periportal hepatocytes also appeared normal with roundednuclei and distinct cytoplasm. DPMT‐treated animals at 0.2 and0.4 mmol kg−1 (data not shown) did not show any noticeablechanges in liver morphology compared with corn oil controlanimals. At the 0.6 mmol kg−1 DPMT dose, sections showedsome damage near the centrilobular vein (data not shown).Cytoplasm in the cells was condensed and irregularly stained.Sinusoids showed more blood than the controls and someleakage of inflammatory cells. Liver sections from rats thatreceived 1.0 mmol kg−1 DPMT exhibited more damagedhepatocytes (Fig. 3B). Vacuolization was noted in the cytoplasmgiving it a lacey appearance. Swollen hepatocytes were evident.Nuclei were also not as round and distinct as in corn oil controls,although no necrotic lesions were visible. DCTA treatment didnot produce any significant changes at the two lower dosesinvestigated (0.2 and 0.4 mmol kg−1, data not shown). Sectionsappeared normal with healthy looking hepatocytes radiatingout of the central vein. Also, no changes were observed inperiportal hepatocytes and the bile ducts were normal. At the0.6 mmol kg−1 DCTA dosage nuclear condensation wasapparent in some hepatocytes (Fig. 3C). Apart from someswollen hepatocytes and narrowed sinusoidal spaces, liversections from 4‐DCTD‐treated animals showed no changes in


hepatic morphology at any dose (1.0 mmol kg−1 shown inFig. 3D) compared with corn oil‐treated controls. Liver sectionsfrom the 2‐DCTD treatment groups (0.2, 0.4 and 0.6 mmol kg−1,data not shown; 1.0 mmol kg−1, Fig. 3E) were generally normal,although some nuclear condensation was evident.BUN levels, urine volume, urine protein and kidney weights

were determined to assess the potential nephrotoxicity of thecompounds (Table 1). BUN concentrations were slightlyelevated with 4‐DCTD treatment at 0.6 and 1.0 mmol kg−1 only.Compared with corn oil and the 0.2 mmol kg−1 dose, DCTAtreatment at 0.4 and 0.6 mmol kg−1 also produced a significantincrease (about 3‐fold) in serum BUN concentrations. In contrast,treatment with DPMT and 2‐DCTD did not produce anysignificant changes in BUN levels. Urine protein levels werenot significantly elevated by any treatment at any of the dosesthat were administered. Diuresis (approximately 3.5‐fold in-crease) was noted with 4‐DCTD treatment at all doses comparedwith controls, but was not dose‐dependent. An increase in urinevolume (about 3‐fold increase) was also noted with DPMTtreatment at 0.6 and 1.0 mmol kg−1 dosages. The increases inurine volumes seen with DCTA and 2‐DCTD were not statisticallysignificant. A slight increase in kidney weight was noted withDCTA treatment at 0.6 and 1.0 mmol kg−1 treatment; however,none of the other treatments produced any significant changesin this parameter (Table 1).When compared with the corn oil controls, DPMT‐ and DCTA‐

treated animals did not exhibit obvious changes in kidneymorphology at any doses (data not shown). Minor swelling inthe proximal tubular cells and slight dilation of collectingtubules was observed with 4‐DCTD, which could havecontributed to the increased urinary flow (data not shown). Incontrast, 2‐DCTD treatment did not produce any noticeablechanges in the kidney morphology at any of the doses that wereevaluated (data not shown).Log P values were calculated as an estimate of the

lipophilicity of the compounds. For the TZD ring modifiedanalogs of DCPT, the relative ranking (most to least lipophilic,Fig. 1) was: DPMT > 2‐DCTD > 4‐DCTD > DCTA.

Phenyl Ring‐modified Compounds

Serum ALT results for this series of compounds are summarizedin Fig. 4. Except for the lowest dose, treatment with thedimethyl analog, DMPT, resulted in marked increases in serumALT levels when compared with the corn oil controls. ALT valueswere significantly elevated with CPTD treatment at all fourdoses. At 1.0 mmol kg−1, both DMPT and CPTD producedsignificant elevations in ALT values when compared with thethree lower doses of each compound. The lowest dose of PTZD(0.2 mmol kg−1) did not produce a significant change comparedwith the corn oil controls, but ALT levels were elevatedapproximately 7‐, 26‐ and 33‐fold at the 0.4, 0.6, and1.0 mmol kg−1 PTZD doses, respectively. With this compound,ALTs were significantly elevated at 0.6 and 1.0 mmol kg−1

compared with the two lower doses. By contrast, serum ALTlevels were normal in rats that received DFMPT at all four doses.Compared with controls, a slight, but statistically significantincrease in liver weights was observed in animals treated withDMPT (0.4, 0.6 and 1.0 mmol kg−1), CPTD (0.6 and1.0 mmol kg−1), and PTZD and DFMPT (1.0 mmol kg−1 dose,Table 2). Liver weights in rats that received DMPT at the


3

Figure 4. Effect of phenyl ring modifications on serum alanine aminotransferase (ALT) levels in rats. Compounds were administered i.p. in corn oil.Controls received corn oil only (4 ml kg−1). Values are means ± SE (N=3–4). An asterisk indicates that the value is significantly different (P<0.05) fromthe corn oil control. Different letters within a treatment group indicate that the doses are significantly different (P<0.05).

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0.2 mmol kg−1 dose were significantly lower than those seenwith the higher doses.

Representative photomicrographs of liver sections from theanimals treated with DMPT, CPTD, PTZD and DFMPT

Table 2. Effect of phenyl ring ring modifications on blood urea nweights in rats

Treatment Dose(mg kg−1)

Blood urea nitrogen(mg dl−1)

Twenty‐four‐urine volu

(ml)

Corn oil — 20.2 ± 2.0 3.7 ± 0.5

DMPT 0.2 18.1 ± 1.3a 5.8 ± 1.4*0.4 22.2 ± 0.4a 9.9 ± 2.2*0.6 27.5 ± 1.4*,b 8.9 ± 1.8*1.0 32.8 ± 3.1*,b 11.3 ± 2.5*

CPTD 0.2 37.9 ± 0.6*,c 5.7 ± 1.5b

0.4 38.1 ± 1.7*,c 12.4 ± 4.6*0.6 40.4 ± 2.7*,c 16.6 ± 0.8*1.0 34.9 ± 1.8*,c 15.3 ± 2.7*

PTZD 0.2 24.8 ± 1.9d 6.1 ± 1.7d

0.4 24.1 ± 0.9d 11.5 ± 3.2d

0.6 28.6 ± 2.0*,d 12.5 ± 2.4d

1.0 31.3 ± 2.4*,d 22.6 ± 0.4*

DFMPT 0.2 19.6 ± 0.5e 10.1 ± 1.3*0.4 21.7 ± 0.4e 14.1 ± 2.6*0.6 22.5 ± 0.5e 20.0 ± 3.1*1.0 32.7 ± 1.4*,f 23.5 ± 1.8*

Compounds were administered i.p. in corn oil. Controls receivedasterisk indicates that the value is significantly different (P<0.05)group indicate that the doses are significantly different (P< 0.05).


(1.0 mmol kg−1 doses) are shown in Fig. 5. Corn oil (4 ml kg−1)is included again for comparison (Fig. 5A). Liver sections fromDMPT‐treated animals at 0.2 and 0.4 mmol kg−1 doses (data notshown) exhibited necrotic lesions with infiltration of inflamma-

itrogen, urine volume and protein content, or liver and kidney

hourme

Six‐hour urineprotein content

(mg)

Liver weight(g per 100%

b.w.)

Kidney weight(g per 100%

b.w.)

1.5 ± 0.4 3.49 ± 0.12 0.37 ± 0.01

,a 0.8 ± 0.3a 3.47 ± 0.20a 0.38 ± 0.01a,a 1.4 ± 0.1a 4.10 ± 0.20*,b 0.40 ± 0.02a,a 1.9 ± 0.2a 4.20 ± 0.05*,b 0.39 ± 0.00a,a 2.8 ± 0.4*,b 3.97 ± 0.04*,b 0.38 ± 0.01a

2.0 ± 0.1c 3.62 ± 0.16c 0.39 ± 0.02b,c 1.9 ± 0.1c 3.84 ± 0.07c 0.41 ± 0.01b,c 2.0 ± 0.1c 4.36 ± 0.06*,d 0.46 ± 0.02*,c,c 1.9 ± 0.1c 3.98 ± 0.14*,c 0.51 ± 0.04*,c

2.1 ± 0.5d 3.69 ± 0.25e 0.36 ± 0.01d

2.1 ± 0.2d 3.68 ± 0.18e 0.40 ± 0.00*,e

3.4 ± 0.9d 4.08 ± 0.08e 0.41 ± 0.01*,e,e 2.3 ± 0.6d 4.24 ± 0.06*,e 0.47 ± 0.01*,f

,f 1.1 ± 0.3e 3.82 ± 0.12f 0.40 ± 0.01 g

,f,g 1.6 ± 0.2e 3.59 ± 0.10f 0.42 ± 0.03 g

,g,h 1.5 ± 0.2e 3.78 ± 0.07f 0.30 ± 0.09 g

,h 1.0 ± 0.2e 4.05 ± 0.15*,f 0.48 ± 0.01*,h

corn oil only (4 ml kg−1). Values are means ± SE (N=3–4). Anfrom the corn oil control. Different letters within a treatment


Figure 5. Effect of phenyl ringmodifications on rat hepatic morphology.Representative photomicrographs (A, corn oil; B, DMPT; C, CPTD; D, PTZD;E, DFMPT) are shown for the highest dose administered for eachcompound (1.0 mmol kg−1; i.p. in corn oil). Controls received corn oil only(4 ml kg−1). White arrowheads define regions of necrosis. Inflammatorycells are indicated by black arrowheads. Magnification is 400×.


11

tory cells, neutrophils and lymphocytes. The damage was morescattered and not limited to the centrilobular region. At higherdoses, 0.6 mmol kg−1 (data not shown) and 1.0 mmol kg−1

(Fig. 5B), necrotic lesions were widespread with many inflam-matory cells seen around the lesions. Typical signs ofliquefactive necrosis were evident, such as cell lysis anddigestion of cellular material by proteolytic enzymes thatproduced holes. Sections from CPTD‐treated animals exhibitednecrotic lesions with acute inflammatory response at all doses.The origin of these lesions was not very clear and normalorganization of cells was lost. Signs of coagulative necrosis, suchas denaturation of proteins and loss of cytoplasmic materialwith preservation of cell outlines were evident in animals atlower doses (data not shown). At 1.0 mmol kg−1 CPTD (Fig. 5C),liver sections showed signs of liquefactive necrosis. The necroticlesions were widespread with infiltration of neutrophils andlymphocytes. Blood was also present at the site of lesions with acomplete loss of normal cellular arrangement. PTZD‐treatedanimals did not show any signs of hepatic damage at the0.2 mmol kg−1 dose (data not shown). At the 0.4 mmol kg−1

dose (data not shown) cells had begun to show some damagewith granular and irregularly stained cytoplasm and indistinctnuclei. With increasing dose, more noticeable damage was seen.At the highest dose (1.0 mmol kg−1, Fig. 5D), larger necrotic


lesions were present and an acute inflammatory responseoccurred. Liver sections from DFMPT‐treated animals did notshow any noticeable changes in morphology at all doses (lowerdoses, data not shown; 1.0 mmol kg−1 dose, Fig. 5E). Sectionsappeared normal and healthy with hepatocytes arranged inrow‐like fashion radiating out of central vein. The bile ductswere intact and also the periportal hepatocytes exhibited nosignificant changes.Compared with corn oil, DMPT and PTZD treatments produced

a statistically significant, but small elevation in BUN levels at thetwo highest doses (Table 2). Animals treated with CPTD exhibiteda statistically significant, but modest (1.8‐fold) increase in BUNlevels at all dosages compared with the corn oil controls. DFPMTtreatment also produced a significant elevation in BUN concen-tration at the highest dosage (1.0 mmol kg−1) compared withcontrols and the three lower doses. A significant increase in urineprotein was only observed with the highest dose of DMPT(Table 2). Diuresis was seen after administration of DMPT andDFMPT at all doses compared with controls and was especiallypronounced at the highest doses of the latter compound(Table 2). An increase in urine volume was also noted with PTZD(1.0 mmol kg−1) and CPTD (0.4, 0.6 and 1.0 mmol kg−1)treatments. Kidney weights were not significantly altered byDMPT treatment at any doses compared with the corn oil‐treatedanimals or to each other (Table 2). However, a small increase wasnoted with CPTD (0.6 and 1.0 mmol kg−1), PTZD (0.4, 0.6 and1.0 mmol kg−1) and DFMPT (1.0 mmol kg−1) treatments.Renal morphology was normal in the DMPT treatment groups

(data not shown). Kidney sections from rats that were adminis-tered CPTD and DFMPT exhibited minor swelling in someproximal tubular cells (data not shown). Collecting tubules wereslightly dilated which could have contributed to the increasedurinary flow. PTZD‐treated rats exhibited dilation of distal tubulesand collecting tubules (data not shown). Overall, only relativelymild kidney damage was seen with all phenyl ring analogs.Based on log P values (Fig. 1), the relative order of lipophilicity

for the phenyl ring‐modified DCPT analogs was (most to leastlipophilic): DFMPT > DMPT > CPTD > PTZD.

DISCUSSION

The glitazones do not cause liver damage in commonlaboratory animal species (Chojkier, 2005) and are not suitablefor evaluating mechanism of toxicity for TZD ring‐containingcompounds in vivo. More recently, 5‐(4‐methoxybenzylidene)thiazolidine‐2,4‐dione, which has a similar TZD ring substitu-tion pattern as the glitazones, was found to be non‐toxic inmice (Luo et al., 2010). Thus, DCPT, which reproduciblyproduces liver damage in rats, may be a useful compoundto explore the potential hepatotoxicity of TZD ring derivatives(Patel et al., 2008). For example, we obtained ALT values of26.7 ± 6.6, 231.5 ± 50.0, 281.7 ± 103.0 and 506.9 ± 99.4 SF unitsml−1 in male rats at DCPT doses of 0.2, 0.4, 0.6 and1.0 mmol kg−1, respectively. Morphological changes, whichincluded necrosis and inflammation, also worsened withincreasing dosage (Patel et al., 2008). In addition to the TZDring, DCPT contains a meta‐substituted dichlorobenzene (DCB)ring (Fig. 1). Although DCBs have been shown to producehepatic injury in rats (Stine et al., 1991; Valentovic et al., 1993),we do not believe that this structural feature is a critical factorin the liver damage associated with DCPT (Crincoli et al., 2008;


5

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Patel et al., 2008). The experiments described here weredesigned to further explore the effect of structure on DCPT‐induced hepatotoxicity in rats.

In contrast to our previous structure‐activity relationshipstudy (Kennedy et al., 2003), all of the analogs described hereincontain a sulfur atom, which is a potential site for oxidativemetabolism. In fact, CYP3A‐mediated TZD ring sulfoxidation wasproposed as an initial step in the generation of reactiveintermediates from the glitazones (Kassahun et al., 2001; Tetteyet al., 2001; He et al., 2004; Baughman et al., 2005). Furthermore,we found that DCPT‐induced hepatotoxicity was CYP3A‐dependent in rats (Crincoli et al., 2008). Generation of asulfoxide may therefore also be necessary for the conversion ofDCPT into a putative toxic species. We observed here thatattachment of a methyl group to the TZD ring (i.e. DPMT, Fig. 1)resulted in a compound that was hepatotoxic. Since thissubstituent should sterically hinder biotransformation at themethylene carbon atom in DPMT, we believe that this positionis not a primary site for metabolic activation in this compound.However, oxidation on the sulfur atom of DPMT is still apossibility, which could account for its toxicity.

A modest elevation in ALT levels was observed with the ring‐opened compound DCTA (Fig. 1, a thioglycolic acid derivative),but no evidence of significant morphological damage was seenin the rat liver sections. DCTA is the only analog that contains anionizable functional group. As an aliphatic carboxylic acid itshould be extensively ionized in plasma. Hence, reduceddistribution into the liver may be a limiting factor for DCTAhepatotoxicity, compared with the other non‐ionizable, neutralcompounds. One possible explanation for the systemic effectsthat we observed with DCTA could be degradation to 3,5‐dichlorophenyl isocyanate. If this potentially reactive compoundwas generated in vivo, protein carbamoylation and toxicitycould result (Brown et al., 1987). Although we have no directevidence that this occurred in the rats with DCTA, thestructurally analogous compound S‐(methylaminocarbonyl)thio-glycolic acid was reported to undergo base‐catalyzed decom-position to methyl isocyanate (Machacek et al., 1981).

4‐DCTD and 2‐DCTD (Fig. 1) were synthesized and tested toaddress the impact of replacing the unsymmetrical TZD ring ofDCPT with regioisomeric thiazolidinone rings. These compoundsdid not produce any significant liver damage at any dose, whichsuggests that DCPT metabolic activation is dependent on thepresence of both carbonyl groups in the TZD ring. The lack ofhepatic injury from these two compounds further suggests thatthe DCB ring is not contributing to hepatotoxicity in our ratmodel. Also, 4‐DCTD and 2‐DCTD probably do not undergoextensive biotransformation to DCPT in vivo (i.e. the thiazoli‐dinone ring in each compound is not metabolized to a TZDring), otherwise liver damage should have been observed.

Lipophilicity can be an important factor in pharmacokinetics(van de Waterbeemd et al., 2001; Giaginis and Tsantili‐Kakoulidou, 2008). Log P values were therefore calculated todetermine the octanol–water partition coefficients (relativelipophilicities) of the DCPT analogs. Based on this analysis, thenon‐toxic analog 2‐DCTD and the toxic compound DPMT havesimilar estimated lipophilicities (log P values = 3.20 and 3.22,respectively). Furthermore, the hepatotoxic ‘parent’ compoundDCPT (Patel et al., 2008) has a similar log P value to the non‐toxicanalog 4‐DCTD (2.73 and 2.71, respectively). These results suggestthat there is no correlation between the lipophilicities of thecompounds and their ability to produce liver damage in rats.


Among the phenyl substituted compounds (Fig. 1), DMPT,CPTD and PTZD produced severe liver damage including largeareas of necrosis with infiltration of neutrophils. Hepatotoxicity,as evidenced by ALT measurements, tended to increase withdose. DFMPT treatment, on the other hand, did not produce anychanges in ALT levels and the histological examination showedno signs of toxicity compared with corn oil controls. These resultsindicate that liver damage is highly dependent on thesubstituents in the phenyl ring and dose. As we observed withthe TZD ring analogs, hepatotoxicity did not correlate withlipophilicity. In fact, PTZD (log P= 1.61), the least lipophiliccompound in this series, produced significant liver damage;whereas the most lipophilic analog DFMPT (log P=3.48) was nothepatotoxic. However, analogs which could release electrons intothe TZD ring via inductive or resonance effects (DMPT and CPTD)were more toxic than the compound that would withdrawelectrons (DFMPT). Conceivably, the phenyl ring substituentscould alter electron density in the TZD ring. This, in turn, couldmodify biotransformation in the TZD ring, which we believe is animportant factor in hepatotoxicity (Crincoli et al., 2008).

We cannot exclude the possibility that other factors, such asalternative biotransformation pathways, could also contribute totoxicity in the phenyl series of compounds. For example, thephenyl ring in DMPT is structurally analogous to 1,3‐dimethylbenzene (m‐xylene). Metabolism of xylenes is believedto occur by an initial benzylic oxidation (Low et al., 1989).Administration of m‐xylene to rats (4.0 mmol kg−1, i.p.) resultedin 45% loss of hepatic glutathione levels within 3 h (van Doornet al., 1980). The same investigators found that 1.3% of a totalm‐xylene dose (3.0 mmol kg−1, i.p.) was excreted in urine asthioether conjugates. These findings suggest that reactiveintermediates can be formed from m‐xylene, although thedoses were 3–4 times higher than we used with DMPT. CPTDcontains a chlorophenyl moiety and, in analogy to chloroben-zene, phenolic metabolites could be formed via epoxideintermediates or direct oxidation (Selander et al., 1975). In fact,chlorobenzene was shown to cause an elevation in serum ALTlevels in rats (Dalich and Larson, 1985); however, the dose usedwas nearly 10‐fold greater than we used with CPTD.

Consistent with our previous findings for DCPT (Kennedy et al.,2003; Patel et al., 2008), both series of analogs produced relativelymild effects on rat kidneys. This is in spite of the fact that DCPT hasclose structural similarity to the nephrotoxicant N‐(3,5‐dichlorophenyl)succinimide (NDPS) (Rankin, 1982). This disparitymay be due to differences in metabolism and distribution of thecompounds. The NDPS succinimide ring undergoes biotransfor-mation in rat liver (Rankin, 2004; Cui et al, 2005). These hepaticmetabolites are then believed to undergo transport through theblood to the kidneys, where theymay accumulate, thereby causingnephrotoxicity. Although biotransformation of the toxic TZDderivatives described herein has not been studied yet, it isconceivable that putative reactivemetabolite(s)may exert damagedirectly in the hepatocytes where they are produced. As notedabove, TZD ring sulfoxidation has been proposed as a possiblebioactivation mechanism for the glitazones (Kassahun et al., 2001;Tettey et al., 2001; He et al., 2004; Baughman et al., 2005).

In conclusion, our findings with the DCPT analogs (TZD ring‐modified series) strongly suggest that an intact TZD ring iscrucial for these compounds to exert liver damage in rats. Webelieve that biotransformation involving the TZD ring sulfuratom may be a factor in hepatotoxicity for the followingreasons: (1) among the TZD ring‐modified compounds, only



DPMT produced significant liver injury (above); (2) DCPT was theonly hepatotoxic analog among a series of compoundscontaining different cyclic imides (i.e. succinimide, oxazolidine-dione, etc.) attached directly to a 1,3‐DCB ring (Kennedy et al.,2003); and (3) CYP inhibitors attenuated DCPT toxicity, whereasa CYP inducer potentiated liver damage in male rats (Crincoliet al., 2008). However, hepatotoxicity is also dependent on thenature of the substituents in the phenyl ring and may be due todifferences in biotransformation among these compounds.

Acknowledgments

The authors would like to thank Dr Joan B. Tarloff, USPDepartment of Pharmaceutical Sciences, for her assistance withthe cardiac punctures. This publication was made possible bygrant number ES012499 (P.J.H.) from the National Institute ofEnvironmental Health Sciences (NIEHS), NIH. Its contents aresolely the responsibility of the authors and do not necessarilyrepresent the official views of the NIEHS, NIH.

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effect of structural modifications on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced...

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