Differential in vitro hepatotoxicity of troglitazone and rosiglitazone among cryopreserved human...

Differential in vitro hepatotoxicity of troglitazoneand rosiglitazone among cryopreserved human

hepatocytes from 37 donors�

Scott Lloyd *, Michael J. Hayden, Yumiko Sakai,Andrew Fackett, Paul M. Silber, Nicola J. Hewitt, Albert P. Li

In Vitro Technologies, Inc., 1450 South Rolling Road, Baltimore, MD 21227, USA

Abstract

We report here our studies on troglitazone and rosiglitazone cytotoxicity in human

hepatocytes isolated from multiple donors to investigate factors responsible for individual

differences in sensitivity to the known hepatotoxicity of these antidiabetic drugs. Using

cellular adenosine triphosphate (ATP) content as an endpoint, cytotoxicity of both drugs was

evaluated in cryopreserved human hepatocytes from 37 donors. We confirmed reports of

others that troglitazone was cytotoxic to human hepatocytes using cellular ATP content as an

endpoint. In addition, we found that rosiglitazone, although less toxic in the study population,

was cytotoxic to hepatocytes in some donors (EC50B/100 mM). ATP content, 3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) metabolism, depletion of

intracellular glutathione, Alamar Blue metabolism, and neutral red uptake were used as

endpoints in a single donor study using freshly isolated human hepatocytes. Troglitazone

appeared to be more toxic than rosiglitazone by all endpoints. From the demographic data

provided to us for each donor, we were able to establish no direct correlation between

cytotoxicity (expressed as EC50 values) and age, sex, smoking status, or alcohol consumption.

We conclude that troglitazone and rosiglitazone are differentially toxic to human hepatocytes,

and that toxicity may be independent of age, sex, tobacco use, and alcohol use.

# 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Adenosine triphosphate; Hepatocytes; Troglitazone

�Support by In Vitro Technologies, Inc., Baltimore, Maryland. This work was presented, in part, at the

second International Symposium on Mechanisms, Models and Predictions of Idiosyncratic Drug Toxicity,

Atlantic City, New Jersey, June 11�/13, 2001.

* Corresponding author. Tel.: �/1-410-455-1242; fax: �/1-410-455-1245

E-mail address: [email protected] (S. Lloyd).

Chemico-Biological Interactions 142 (2002) 57�/71

www.elsevier.com/locate/chembiont

0009-2797/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.

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1. Introduction

The thiazolidinediones are a relatively new class of drugs used to treat (type 2)

non-insulin dependent diabetes mellitus, or NIDDM ([1]). This novel class of

antidiabetic agents has proven useful as either a monotherapy, or in combination

therapy with insulin, metformin or sulfonylureas in the treatment of NIDDM ([1]).

The thiazolidinediones are selective agonists of the peroxisome proliferator-activated

receptors (PPARs) and have been shown to lower insulin resistance in both diabetic

and non-diabetic individuals ([2,3]). The PPARs, especially PPARg, are cell surface

receptors and transcription factors present in adipocytes, skeletal muscle, and liver

Fig. 1. Dose response curves to troglitazone (triangles) and rosiglitazone (squares) for cryopreserved

hepatocytes from 37 different donors (panels A through KK).

S. Lloyd et al. / Chemico-Biological Interactions 142 (2002) 57�/7158

tissue, and are key elements in the regulation and metabolism of glucose in response

to insulin ([4]). Many patients suffering from NIDDM lack glycemic control due to

non-responsiveness of PPARs, making the use of thiazolidinediones an attractive

therapeutic approach.

Troglitazone was introduced to the market in 1997 and was the first thiazolidi-

nedione to be approved for use in the treatment of NIDDM ([1]). However, during

its first 2 years on the market, a number of cases were reported that associated

troglitazone with acute liver toxicity and failure, leading to the death of patients

using the drug or necessitating liver transplant in some cases ([5,6]). Subsequent

clinical investigations lead to its withdrawal from the UK market in December 1997

and to its withdrawal from the US market in March of 2000 based on data that

associated troglitazone with cases of serious idiosyncratic liver toxicity ([7]).

Fig. 1 (Continued)

S. Lloyd et al. / Chemico-Biological Interactions 142 (2002) 57�/71 59

Rosiglitazone, a relatively non-toxic alternative to troglitazone, was introduced to

the market in 1999 ([1]). While the incidence of significant hepatotoxicity caused by

rosiglitazone has not been observed in controlled clinical trials, two cases have been

reported which implicate rosiglitazone as a causative agent of idiosyncratic drug

toxicity (IDT) as well ([8�/10]). Several studies have proposed a variety of

mechanisms of troglitazone cytotoxicity, including parent compound toxicity in

human hepatocytes, the formation of reactive intermediates and glutathione (GSH)

conjugates in human liver microsomes, and both cholestatic and apoptotic potentials

in cultured rat hepatocytes ([11,12]). Mechanisms of hepatocellular injury caused by

rosiglitazone have not been well studied, likely because the causal relationship

between the drug and hepatotoxicity remains uncertain. This study used a panel of

cryopreserved human hepatocytes isolated from demographically diverse donors,

Fig. 1 (Continued)


and freshly isolated hepatocytes from a single donor, to demonstrate dose-dependent

troglitazone toxicity and to compare individual sensitivities to troglitazone androsiglitazone on the basis of age, sex, alcohol or tobacco use.

2. Materials and methods

2.1. Incubation of hepatocytes with troglitazone and rosiglitazone

Cryopreserved hepatocytes from 37 individual donors, along with freshly isolated

hepatocytes from a single donor, were used in this study. The cells were isolated,

Fig. 1 (Continued)


cryopreserved, and thawed using previously published procedures ([13�/15]).

Troglitazone and rosiglitazone were obtained from Parke-Davis (Ann Arbor, MI)

in powder form and prepared as a 2000�/stock solution in dimethyl sulphoxide

(DMSO; Sigma, St. Louis, MO), then diluted to a 2�/concentrated dosing solution

in Dulbecco’s Modified Eagle Medium (Sigma). Cytotoxicity assays were initiated by

the addition of an equal volume of cryopreserved hepatocyte suspension to the

media containing troglitazone or rosiglitazone. The final troglitazone and rosiglita-

zone concentrations used in the incubations were 10, 20, 50, 100 and 200 mM. The

final DMSO concentration was 0.1% (v/v). In a single related experiment, freshly

isolated hepatocytes were incubated overnight on collagen (Cohesion, Palo Alto,

CA) coated 96 well plates. The plating media was then replaced with incubation

Fig. 1 (Continued)


Fig. 1 (Continued)


media containing 10, 20, 50, 100 or 200 mM troglitazone or rosiglitazone for a 24-h

incubation period. Incubations of hepatocytes with drug and control solutions were

carried out in a cell culture incubator maintained at 37 8C, 5% CO2 in saturating

humidity. The treatment duration was 2 h for cryopreserved hepatocytes.

2.2. Cell viability analysis

ATP content analysis was performed using the ATP Lite-M kit from Packard

Marketing (Groningen, The Netherlands). Briefly, the hepatocytes were lysed after

incubation followed by the addition of luciferin and luciferase. After a 10-min dark

adaptation, luciferin luminescence was quantified using a Wallac Victor 2 Micro-

plate Reader (Perkin Elmer, Gaithersburg, MD). ATP standards of known

concentrations were used to convert relative luminescent units readings to ATP

concentrations.

Neutral red uptake analysis was performed using a commercially available neutral

red kit (Sigma). The neutral red dye was added to the incubation media for 3 h at

37 8C, 5% CO2. The dye was fixed and solublized, and the amount of neutral red

taken up by the hepatocytes was measured photometrically.

MTT metabolism analysis was performed using a commercially available MTT

reagent (Sigma). The reagent was added to the incubation media at the end of the

troglitazone or rosiglitazone treatment and incubated for an additional 3 h at 37 8C,

5% CO2. The formazan crystals formed were dissolved in acidified isopropanol and

measured photometrically following overnight incubation at 4 8C.

Alamar Blue was purchased from Bio Source International (Camarillo, CA) and

added to the incubation media for 3 h at 37 8C, 5% CO2, and its reduction was

measured fluorometrically.

GSH content was measured using a modified version of previously published

methods ([16]) based on the reaction of GSH with o -pthaldehyde (Sigma).

Fluorescent emission values for the GSH assay were converted to micromolar

GSH concentrations using a standard curve based on the incubation of o-

pthaldehyde with known concentrations of glutathione (Sigma).

2.3. Data analysis

EC50 values for troglitazone and rosiglitazone, and associated statistical analyses

(Student’s t -test) were generated using Graph Pad (Prism Computer Software, San

Diego, CA). Least squares regression and other statistical analyses between donor

properties and EC50 values were investigated using Microsoft Excel.


3. Results

3.1. Troglitazone and rosiglitazone cytotoxicity

Troglitazone or rosiglitazone cytotoxicity was evaluated in cryopreserved human

hepatocytes from 37 donors using ATP content as an endpoint following 2 h

treatment with either drug (Fig. 1). Troglitazone was found to be cytotoxic (EC50B/

100 mM) in 86% (32 of 37) of the donors screened, while rosiglitazone appeared to be

cytotoxic in 19% (7 of 37) of the donors (Table 1). In one case, the EC50 value fell

below 50 mM for troglitazone (Lot 109, Table 1). Cytotoxicity of both drugs was also

measured in freshly isolated human hepatocytes following a 24 h incubation using

ATP content, MTT metabolism, GSH depletion, Alamar Blue metabolism and

neutral red uptake as endpoints (Fig. 2). Troglitazone was found to be cytotoxic

(EC50B/100 mM) to fresh hepatocytes by all endpoints except GSH depletion, while

rosigliatazone was not found to be cytotoxic by any of the endpoints considered.While some depletion of GSH by both drugs was observed in fresh hepatocytes,

neither drug caused GSH levels to fall below 70% of vehicle control.

3.2. Correlation of age, sex, and environmental factors with sensitivity to troglitazone

or rosiglitazone

The demographic information for the 37 cryopreserved hepatocyte donors used

are shown in Table 1. No apparent correlation was observed between EC50 values oftroglitazone (Fig. 3) or rosiglitazone (Fig. 4) and age, sex, tobacco use or alcohol

consumption.

4. Discussion

IDT (adverse drug reactions which occur at low frequency in the human

population) is a major challenge in drug development. Current pre-clinical andclinical safety trials appear to be inadequate predictors of IDT. One approach to

understanding IDT is to elucidate the key properties responsible for the observed

toxicity with the aim of identifying susceptible human subpopulations.

Troglitazone was removed from the market as a result of its association with IDT

in the clinical setting ([1]). Rosiglitazone, which has been associated with IDT in only

two isolated cases worldwide, is currently on the market as a replacement drug of the

same class ([8]). In the clinical setting, the differences in toxicity may due in part to

the fact that troglitazone is administered at a relatively high dose compared torosiglitazone. The clinical dose difference between these two drugs may relate to why

the EC50 values in Table 1 for rosiglitazone are no more than two times the values

for troglitazone in most donors, since the same doses of each drug were used in vitro.

We evaluated troglitazone and rosiglitazone cytotoxicity in cryopreserved human

hepatocytes from multiple donors as part of our research program to develop

approaches to predict IDT. One purpose of this study was to evaluate and compare


Table 1

Background and demographic information on cryopreserved human hepatocyte donors screened for sensitivity to troglitazone and rosiglitazone with EC50

values from ATP assays

Lot Rosi Trog Race Age Sex Weight

(kg)

Smoker Alcohol

use

Medications/drugs Disease history

EC50 EC50

59 101.7 92.6 Caucasian 33 Male 75 Yes Social Occasional marijuana as teen,

hydrocordone

Childhood asthma

61 62.6 63.7 Caucasian 38 Male 95 Yes Daily No No

62 73.1 63.6 Caucasian 46 Male 102 No Daily Marijuana No

66 146.2 95.1 Caucasian 48 Male 96 No Non-

drinker

Agent orange exposure Heart disease

70 95.3 52 Caucasian 57 Male 113 Yes Social Occasional marijuana Hypertension, 15�/20 yrs

71 103.2 83.4 Hispanic 23 Male 91 No Social Xanax (not prescribed) Childhood asthma

75 188.6 92.5 Caucasian 15

months

Male 10 No Non-

drinker

No No

83 128.4 92.1 Black 56 Male 89 No Non-

drinker

N/A Hypertension

86 135.9 76.3 Caucasian 73 Female 51 No Non-

drinker

No Anemia 1 yr ago


drinker

No No

89 128.4 92.1 Caucasian 44 Female 85 Yes N/A N/A N/A

90 162.2 78 Caucasian 51 Female N/A Yes Social N/A Chronic cough, kidney infection

91 �/500 139.4 Caucasian 72 Female 60 Yes Non-

drinker

Nitropaste Emphysema, mild arthritis, myocar-

dial infarction (15�/17 yrs ago)

95 138.8 64.9 Caucasian 43 Male 170 No N/A Occasional marijuana N/A

105 87.4 67.1 Caucasian 59 Male 111 Yes Social Cardizem, zocor Heart disease, hypertension

108 106.3 105 Asian 59 Male 64 No Daily N/A Hemia, hypertension

109 128.6 20.6 Caucasian 69 Male 88 No Social N/A NIDDM (10 yrs), hypertension, high

cholesterol transitional blood cancer

111 88.8 55.7 Black 59 Female 52 Yes Social Diazide Hypertension

113 132.2 91.4 Caucasian 61 Female 50 Yes Social No N/A

114 88.1 82.7 Caucasian 47 Female 100 No Social Opiates N/A

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Table 1 (Continued )

Lot Rosi Trog Race Age Sex Weight

(kg)

Smoker Alcohol

use

Medications/drugs Disease history

EC50 EC50

118 233.8 142.8 Caucasian 48 Female 57 Yes Daily N/A N/A

119 191.4 90.7 Caucasian 48 Female 57 Yes Daily N/A N/A

122 124.4 71.9 Hispanic 42 Male 177 No Daily Some marijuana Hypertension (untreated)

129 196.4 92.2 N/A N/A N/A N/A N/A N/A N/A N/A


drinker

No No

133 125.3 81.5 Hispanic 59 Female 79 Yes Social Provitol, quanarin, monopril,-

celexia, flovent, deserel

Depression

CNV 133.8 93.2 Caucasian 38 Male 103 Yes Social Catenolol Hypertension (B/5 yrs), hepatitis C in

late 1980s

DRL 103.8 78.1 Caucasian 44 Male 100 Yes Social No N/A

EFA 78.9 63.2 Caucasian 16 Male 73 No Non-

drinker

No No

ETR 117.9 85.6 Caucasian 51 Female 74 No Non-

drinker

No No

EVY 105.8 69.3 Caucasian 63 Male 124 No Daily Cumadin, prinavil, lassix,digi-

tech

N/A

GNG 105.4 85.3 Caucasian 45 Female 84 Yes Social No No

HRK 124.5 115.2 Caucasian 66 Female 90 No Non-

drinker

25 yrs (for hypertension) Hypertension

KMI 103.7 101.3 Caucasian 45 Male 110 No Social No Hypertension

MQF 123.3 57.8 Caucasian 49 Female 104 No Social N/A N/A

MYO �/500 62.6 Caucasian 59 Female 90 No Non-

drinker

No Adult onset diabetes

TVC 74.4 91.7 Caucasian 58 Female 55 Yes Non-

drinker

Provacol, claritan, plavix, cli-

mare, lopressor

Vascular disease

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the potential hepatotoxic effects of these two closely related drugs among a large

panel of individuals with diverse demographic backgrounds using an in vitro

approach. This was done in an effort to assess the sensitivity of our pre-clinical in

vitro model system by determining the cytotoxic effects of a known causative agent

of IDT in comparison with a structurally similar compound not associated with

IDT. Our results correlate well with the clinical findings associated with the

thiazolidinediones. Troglitazone was found to be cytotoxic (EC50B/100 mM) at a

rate of 86% in our in vitro study. Our results suggest that cryopreserved human

hepatocytes provide a good pre-clinical in vitro model system for the evaluation of

potential causation of IDT in the early stages of drug development. In addition, the

use of freshly isolated hepatocytes in such studies allows for toxicity evaluation

Fig. 2. Dose response curves to troglitazone (triangles) or rosiglitazone (squares) for freshly isolated

primary hepatocytes from a single donor as evaluated by ATP production (panel A), MTT metabolism

(panel B), GSH depletion (panel C), Alamar Blue metabolism (panel D) or Neutral Red uptake (panel E).


following extended incubation periods. However, the availability of fresh tissue

imposes limits on the number of donors that can be included in such studies. Finally,

the use of multiple endpoints may provide insights into the mechanisms of IDT and

help to eliminate experimental artifacts specific to a single endpoint.A secondary purpose of the study was to clarify relationships between troglitazone

or rosiglitazone cytotoxicity and donor age, sex, alcohol consumption, and tobacco

use, while comparing the observed in vitro hepatotoxic effects of these two related

drugs. We observed no apparent correlation between cytotoxicity and the donor

properties considered for either drug. It is important to note, however, that the

availability of demographic data specific to each donor empowers researches with

the ability to establish relationships between specific donor attributes and IDT.

In summary, our results provide evidence for the usefulness of cryopreserved

hepatocytes in the evaluation of drug toxicity in vitro. The results of the study relate

well to the clinical findings that troglitazone is more toxic than rosiglitazone to the

liver. However, a significant percentage of individuals were also found to be sensitive

to rosiglitazone in our study, suggesting the need for further clinical evaluation or, at

Fig. 3. Correlation of troglitazone hepatotoxicity with donor age (panel A), sex (panel B), smoking status

(panel C) or level of alcohol consumption (panel D).


a minimum, the close and careful monitoring of liver function in patients receiving

this compound for the treatment of type 2 diabetes.

Acknowledgements

Presley Mozone, In Vitro Technologies, Inc., for assistance with hepatocyte

handling and cryostorage. Danielle F. Hill, In Vitro Technologies, Inc., for

assistance with hepatocyte handling and cryostorage. Blaise Considine, In Vitro

Technologies, Inc., for assistance with manuscript preparation.

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