Structure–activity relationships for thiol reactivity and rat or human hepatocyte toxicity induced...

608 K. CHAN ET AL.

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 608–620

DOI: 10.1002/jat

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2008; 28: 608–620Published online 2 November 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jat.1312

Structure–activity relationships for thiol reactivity andrat or human hepatocyte toxicity induced by substitutedp-benzoquinone compounds

Katie Chan,1,* Neil Jensen2 and Peter J. O’Brien1

1 University of Toronto, Faculty of Pharmacy, Toronto, ON, Canada M5S 2S22 Celsis-In Vitro Technologies, 1450 South Rolling Road, Baltimore, MD 21227, USA

Received 3 August 2007; Revised 7 September 2007; Accepted 13 September 2007

ABSTRACT: Covalent binding of toxic chemicals to cellular targets is a molecular interaction that initiates a wide

array of adverse biological effects. The creation of a covalent bond can be cited as a key initiating step along many toxi-

city pathways which must be predicted in order to predict the potential of a chemical to cause specific harmful effects.

Currently, quantitative structure-activity relationship (QSAR) models are being improved by focusing on endpoints such

as simple electrophile reactivity for covalent interactions rather than on commonly used complex toxicity endpoints. The

cytotoxicity and electrophilic reactivity of 10 p-substituted benzoquinone derivatives, which are well known electrophilic

alkylating agents, were investigated under the premise that QSAR toxicity models can be improved when the molecular

triggering event is considered. Hepatocyte toxicity was determined by incubation of individual compounds with freshly

isolated rat or cryopreserved human hepatocyte suspensions. The potential for chemical reactivity between a chemical and

cellular target was measured by determining non-enzymic reactivity with glutathione, representing thiol nucleophiles.

The decline in free thiol moieties was measured to characterize the electrophile reactivity. It was found that the degree

of rat hepatotoxicity induced by benzoquinones correlated with the rate at which they non-enzymically react with

glutathione and to various global and atomic electronic frontier orbital parameters which described electrophilicity.

Human hepatocytes showed similar results but the statistical significance was much lower. The QSAR expressions

suggest that covalent binding reactivity serves as a good correlate to hepatotoxicity and could improve QSAR modeling

for potential toxicity risks. Copyright © 2007 John Wiley & Sons, Ltd.

KEY WORDS: hepatocytes; QSAR; covalent binding; glutathione; cytotoxicity; benzoquinones; cryopreserved human hepatocytes;

chemical reactivity

* Correspondence to: Katie Chan, University of Toronto, Faculty of Phar-

macy, Toronto, ON, Canada M5S 2S2.

E-mail: [email protected]

for biological activity. Modeling of adverse outcomes in

the past has been a challenge because chemicals from

all different structural classes cause similar biological

effects, while also having multiple pathways that lead

to these same events. A new approach has been initiated

to improve the strategy behind QSAR modeling. This in-

volves a shift from modeling complex toxicity endpoints,

such as organ toxicity, skin irritation/sensitization,

mutagenicity and carcinogenicity, to a central event that

is common to these various toxicological events. Protein

covalent binding, which is the adduction of reactive

metabolites to critical proteins, is considered to be a key

initiating step that can be used to predict the potential

for a chemical to cause specific harmful effects (Schultz

et al., 2006). For example, liver toxicity induced by many

xenobiotics can be attributed to their electrophilic

metabolites reacting with critical cellular nucleophiles

(proteins and DNA), forming an irreversible covalent

bond that can lead to adverse events such as cell death or

hypersensitivity (Liebler and Guengerich, 2005). QSAR

modeling therefore has been shifted to focus on the event

of covalent binding and the limited chemical reactivities

Introduction

Quinones are a ubiquitous class of compounds which are

common constituents in plants and biologically relevant

molecules (Bolton et al., 2000). Quinone moieties are also

present in many drugs and can be formed as metabolites

from a variety of xenobiotics (Verma, 2006). They repre-

sent structural features which have the potential to create

a variety of hazardous effects in vivo, as they are highly

reactive electrophiles and can bind covalently to critical

proteins and DNA causing toxicity (Rossi et al., 1986;

Tapper et al., 2000; van Ommen et al., 1988).

Quantitative Structure-Activity Relationship (QSAR)

modeling is a tool used in toxicity assessments that

attempts to correlate biological activity with structural

features of molecules. This is based on the general

premise that molecular properties that are characteristic of

all active compounds must in some way be essential

STRUCTURE-ACTIVITY RELATIONSHIPS FOR THIOL REACTIVITY 609


DOI: 10.1002/jat

Table 1. Structure of p-benzoquinone derivatives

Substituents at position: C′p-Benzoquinone Abbreviation 2 3 5 6

1 chloroanil -Cl4 Cl Cl Cl Cl

2 2,5-dichloro-benzoquinone -2,5-Cl2 Cl H Cl H

3 2-bromo-benzoquinone -2Br Br H H H

4 2-tert butyl-benzoquinone -2(CH3)4 (CH3)4 H H H

5 2-methyl-benzoquinone -2CH3 CH3 H H H

6 p-benzoquinone -none H H H H

7 2,6-dimethyl-benzoquinone -2,6-(CH3)2 CH3 H H CH3

8 2,6-dimethoxy-benzoquinone -2,6-(OCH3)2 OCH3 H H OCH3

9 2,3,5-trimethyl-benzoquinone -2,3,5-(CH3)3 CH3 CH3 CH3 H

10 2,3,5,6-tetramethyl-benzoquinone -2,3,5,6-(CH3)4 CH3 CH3 CH3 CH3

that drive this event (Aptula et al., 2005; Aptula and

Roberts, 2006). A method for modeling the reactivity of

electrophiles has been developed using glutathione (GSH)

as a surrogate for soft nucleophiles. Schultz et al. pro-

posed that this model will simulate the relative rates at

which a reactive chemical is likely to bind protein targets

and provide a measure of the inherent risk of reactivity

(Schultz et al., 2005). Using this method in combination

with QSAR modeling (to identify structural indicators of

electrophilic reactivity), is proposed as an alternative high

throughput approach to determining the potential toxicity

of chemicals.

This study investigated the cytotoxicity and electrophi-

lic reactivity of ten p-benzoquinone derivatives (Table 1).

Cytotoxicity was determined in both rat and human

hepatocytes. Electrophilic reactivity was measured using a

non-enzymic thiol assay based on GSH. Structure-activity

relationship models were derived to determine the physico-

chemical parameters that underlie p-benzoquinone alkylat-

ing activities and toxicity. The goal was to determine the

key structural features which impact reactivity and whether

intrinsic reactivity and potential for covalent binding can

serve as a risk indicator for hepatotoxicity. This may play a

significant role in improving future toxicological evaluation.

Materials and Methods

Chemicals

All chemicals were of the highest commercial grade avail-

able. Chloranil, 2,5-dichlorobenzoquinone, 2-tert-butyl

benzoquinone, 2-methylbenzoquinone, benzoquinone,

2,6-dimethylbenzoquinone, 2,6-dimethoxybenzoquinone,

2,3,5,6-tetramethylbenzoquinone, trypan blue, 3-[4,5,-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

(MTT) and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB)

were purchased from Sigma Aldrich Co. Canada Ltd

(Oakville, ON, Canada).

2,3,5-(CH3)3-benzoquinone and 2-bromobenzoquinone

were prepared as described by Rossi et al. (1986). Briefly,

their respective quinols (5% w/v) were oxidized with

Ag2O (5 eq) in dry diethyl ether until complete, followed

by crystallization of the quinone from ether onto fresh

dry Ag2O under a slow stream of helium gas. Quinones

were then sublimed three times to yield brilliant yellow

crystals.

Hepatocytes

Male Sprague-Dawley rats weighing 275–300 g (Charles

River Canada Laboratories, Montreal, QC, Canada) were

housed in ventilated plastic cages over PWI 8-16 hard-

wood bedding. There were 12 air changes per hour, 12 h

light photoperiod (lights on at 0800 h) and an environ-

mental temperature of 21–23 °C with a 50–60% relative

humidity. The animals were fed with a normal standard

chow diet and tap water ad libitum. Fresh hepatocytes

were isolated from rats by collagenase perfusion of the

liver according to the method described by Moldeus and

coworkers (Moldeus et al., 1978). Cells (106 cells ml−1)

(10 ml) were suspended in Krebs-Henseleit buffer (pH 7.4)

containing 12.5 mM HEPES in continually rotating 50 ml

610 K. CHAN ET AL.


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round-bottomed flasks and maintained under an atmo-

sphere of 95% O2 and 5% CO2 in a water bath of 37 °C

throughout the entire experiment. The hepatocytes used

were at least 85% viable immediately after isolation. Fresh

rat hepatocytes were prepared before each experiment.

Cryopreserved human male hepatocytes were obtained

from Celsis-In Vitro Technologies. Briefly, cryopreserved

cells were thawed at 37 °C, and suspended in pre-warmed

(37 °C) InVitroGRO™ CP medium (Celsis-In Vitro Tech-

nologies, Baltimore, MD). The cell suspension was mixed

gently, centrifuged at 50 g for 5 min at 4 °C, and the

supernatant discarded. The cells were then resuspended in

InVitroGRO™ HI medium (Celsis-In Vitro Technologies,

Baltimore, MD). Hepatocytes (0.5 × 106 viable cells ml−1)

(0.2 ml) were suspended in 48-well plates, under 37 °C

and 5% CO2, on a rocking apparatus. Limited to avail-

ability, human hepatocytes used in these experiments

were from one donor (lot. ZAG, see www.celsis.com for

details). The viability of human hepatocytes post-thaw

was 77% ± 2% (viability before cryopreservation, 85%).

An advantage of using one donor in these experiments

was that it avoided variation between cell batches from

different humans. Such variation occurs to a lesser extent

in laboratory cross-bred rats. Both freshly isolated rat and

cryopreserved male hepatocytes were treated with various

doses of p-benzoquinone compounds and incubated for

up to 3 h.

Cell Viability

The viability of freshly isolated hepatocytes was deter-

mined by the trypan blue (0.4% w/v) exclusion test

(Chan et al., 2007). The viability of cryopreserved

hepatocytes was assessed by adding 0.02 ml of a solution

of 3-[4,5,-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium

bromide (MTT, 5 mg ml−1 in H2O) to each well. The

plates were returned to the incubator for another 3 h.

After the incubation 0.04 N acidified isopropanol (0.2 ml)

was added per well to dissolve the MTT formazan. The

absorbance of MTT formazan was measured at 572 nm

and 690 nm. The corrected absorbance was determined

by subtracting the 690 nm value from the 572 nm value

as a background correction. Cytotoxicity was defined

by LC50, the concentration of a chemical (μM) added

to hepatocytes which caused 50% of cells to be dead at

2 h. The results were expressed as the mean ± SEM from

three separate experiments.

Non-enzymatic Glutathione Reactivity (ThiolReactivity)

The non-enzymatic reaction of various electrophiles

with glutathione (GSH) was assayed using DTNB (5,5′-dithio-bis-(2-nitrobenzoic acid)). Stock solutions of GSH

were made fresh in 0.1 M potassium phosphate buffer

containing 1 mM EDTA to prevent oxidation. Stock solu-

tions of electrophile compounds were prepared in DMSO

at 100× their desired concentrations. All reactions took place

at 25 °C in 0.1 M potassium phosphate buffer (pH 8.0).

The final volume of the reaction mixture was 10 ml, to

which 1 ml of GSH stock solution was added with 0.1 ml

of electrophile stock solution. After a given reaction time

ranging from 10 to 180 min, 0.1 ml of a DTNB stock

solution (0.01 g ml−1) dissolved in 1% sodium citrate,

was added to the reaction. The absorbance at 412 nm was

read immediately with a Pharmacia Biotech Ultraspec

1000 spectrophotometer. The optical density of the

chromophore corresponds to the free thiol concentrations

of the solution, i.e. unreacted GSH. To determine the

reactivity of compounds with GSH, pseudo first-order

kinetics was assumed for the decrease of GSH concentra-

tion, with electrophile added in excess. From the loss

of GSH concentration over time, the rate of reactivity

of electrophiles toward GSH, kGSH, was calculated using

the following equations derived by Freidig et al. (1999).

kGSH,obs = (ln CGSH,0 − ln CGSH,t)/t (1)

kGSH = kGSH,obs/CEl,0 (2)

where, CGSH,0 is the initial concentration of GSH, CGSH,t is

the final concentration of GSH, t is the reaction incu-

bation time, and CEl,0 is the electrophile concentration.

All reaction rates were measured with a control (with

GSH) and a blank (without GSH). No decrease of GSH

was found in control incubations which were spiked with

DMSO only. Negative controls (run without GSH) were

performed to make sure test compounds did not interfere

with absorbance of DTNB-conjugates. The reaction rates

were expressed as the mean ± SEM from three separate

experiments.

Quantitative-Structure Activity Relationships(QSAR) and Calculation of PhysicochemicalParameters

Molecular structures of all p-benzoquinone derivatives

were prepared using CAChe molecular modeling soft-

ware (Version 6.1.12 for Windows 2000/XP, (2000–2003)

Fujitsu Ltd). The physico-chemical parameters used

were restricted to those hypothesized to be associated

with the general cytotoxic and reactivity mechanism

for p-benzoquinones and calculated using the CAChe

MOPAC quantum mechanics application. ELUMO (energy

of the lowest unoccupied molecular orbital), EHOMO

(energy of the highest molecular orbital), dipole moment

(μ), and electron density properties (nucleophilic frontier

density (NufD) and LUMO) for each carbon atom on

the aromatic ring, were calculated using the MOPAC PM5



DOI: 10.1002/jat

semi-empirical Hamiltonian algorithm. Log P and mole-

cular refractivity (MR) were also calculated by CAChe,

using an atom typing scheme of Ghose and Crippen

(Ghose et al., 1988). SPARC On Line Calculator v3.1,

(Sparc OnLine Calculator. http://ibmlc2.chem.uga.edu/

sparc [Jan 2007]) was used to calculate one electron

reduction potentials (E1/2) of the molecules. Statistical

calculations of correlations between physiochemical

parameters and biochemical data were carried out by

multiple linear regression analysis using Sigma Stat

(V2.03, 1992–1997 SPSS Inc.).

Results

Hepatocyte Cytotoxicity

Concentrations of a series of ten p-benzoquinone con-

geners required to cause a 50% decrease in hepatocyte

cell viability (LC50 μM) in 2 h were determined and are

given in Table 2. The viability of control (untreated)

hepatocytes remained constant for both rat and human

hepatocytes during the 2 h incubation and up to as long

as 3 h incubation. At longer times the viability for both

cells in suspension began to decline. The cytotoxic effec-

tiveness of p-benzoquinone congeners towards rat hepato-

cytes ranged from a LC50 of 18 μM to 800 μM, resulting

in a 44-fold difference between the most and least cyto-

toxic p-benzoquinone. Chloranil was the most cytotoxic

benzoquinone and 2,3,5-6-tetramethyl benzoquinone, was

the least toxic. In human hepatocytes, the cytotoxic effec-

tiveness ranged from 80 μM to 1600 μM, resulting in a 20-

fold difference between the most and least cytotoxic

p-benzoquinone. The most cytotoxic p-benzoquinone in

human hepatocytes was 2,6-dimethylbenzoquinone, while

halobenzoquinones were the most cytotoxic towards rat

hepatocytes. The order of cytotoxicity towards rat hepa-

tocytes was: 2,3,5,6-Cl4 > 2,5-Cl2 > 2Br > 2-tbutyl > 2CH3

> BQ > 2,6-(CH3)2 > 2,6-(OCH3)2 > 2,3,5-(CH3)3 > 2,3,5,6-

(CH3)4. The order of cytotoxicity in human hepatocytes,

differed from what was seen with rat hepatocytes, and

was: 2,6-(CH3)2 > 2,6-(OCH3)2 > 2CH3 > 2,5-Cl2 > BQ >2-tbutyl > -Cl4 > 2,3,5-(CH3)3 > 2Br > 2,3,5,6-(CH3)4.

Quantitative Structure Activity Relationships(QSAR) and Cytotoxicity

Rat Hepatocytes

Multiple linear regressions (MLR) analysis was used to

derive structure activity relationships using the cytotoxi-

city data in Table 2 and the physico-chemical descriptors

calculated in Table 3. The derived QSAR models are

Table 2. p-Benzoquinone hepatocyte cytotoxicity and non-enzymic glutathione reactivity

Rat hepatocyte Human hepatocyte Non-enzymictoxicity toxicity glutathione reactivity

p-Benzoquinone LC50 (2 h) μM log LC50 LC50 (2 h) μM log LC50 kGSH [M−1 min−1] log kGSH

1 chloroanil 18 ± 2 1.26 200 ± 20 2.30 4116 ± 440 3.61

2 2,5-dichloro-benzoquinone 23 ± 2 1.36 140 ± 15 2.15 6052 ± 600 3.78

3 2-bromo-benzoquinone 31 ± 5 1.49 500 ± 45 2.70 1435 ± 180 3.16

4 2-tert butyl-benzoquinone 32 ± 3 1.51 160 ± 20 2.20 846 ± 80 2.93

5 2-methyl-benzoquinone 46 ± 5 1.66 117 ± 13 2.07 2423 ± 225 3.38

6 p-benzoquinone 57 ± 6 1.76 143 ± 15 2.16 1712 ± 148 3.23

7 2,6-dimethyl-benzoquinone 80.5 ± 7 1.91 83 ± 10 1.92 1601 ± 177 3.20

8 2,6-dimethoxy-benzoquinone 81.5 ± 8 1.91 100 ± 10 2.00 696 ± 70 2.84

9 2,3,5-trimethyl-benzoquinone 300 ± 35 2.48 233 ± 25 2.37 376 ± 45 2.58

10 2,3,5,6-tetramethyl-benzoquinone 800 ± 77 2.90 1600 ± 100 3.20 113 ± 11 2.05

Table 3. Calculated physico-chemical parameters for p-benzoquinones

ELUMO EHOMO E1/2 Dipole Molecularp-Benzoquinone (eV) (eV) log P (eV) moment (μ) refractivity (MR)

1 chloroanil −2.832 −9.841 2.527 −0.394 0.004 47.00

2 2,5-dichloro-benzoquinone −2.624 −10.289 1.491 −0.23 0.004 37.39

3 2-bromo-benzoquinone −2.46 −10.65 1.247 −0.169 0.682 35.40

4 2-tert butyl-benzoquinone −2.124 −10.702 2.082 −1.177 1.079 46.44

5 2-methyl-benzoquinone −2.204 −10.787 0.922 −1.046 0.749 32.82

6 p-benzoquinone −2.286 −10.921 0.455 −0.984 0.006 27.78

7 2,6-dimethyl-benzoquinone −2.126 −10.727 1.389 −1.099 1.185 37.86

8 2,6-dimethoxy-benzoquinone −2.122 −10.233 −0.05 −0.873 0.322 40.70

9 2,3,5-trimethyl-benzoquinone −2.049 −10.278 1.86 −1.143 0.671 42.90

10 2,3,5,6-tetramethyl-benzoquinone −1.976 −10.253 2.324 −1.182 0.001 47.94

http://ibmlc2.chem.uga.edu/sparc

612 K. CHAN ET AL.


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Figure 1. Plot of log LC50 cytotoxicity values for substituted p-benzoquinones in rat hepatocytes against their ELUMO

values. The line represents Eqn. (1) log LC50 = 24.54 + 17.7 ELUMO + 3.36 (ELUMO)2, n = 9, r2 = 0.90, s = 0.04, F = 25.9,P = 0.002, (�) correlated data; (�) outlier

reported in Table 5. In QSAR studies chemical congeners

contain common templates, which in this study is a rigid

aromatic ring with para substituted carbonyl groups

(Table 1). These carbonyl groups are important as they

identify this benzoquinone group into a specific reaction

mechanism mode. p-Benzoquinones to a greater or lesser

extent act as Michael acceptor electrophiles depending

on the substituents present. Since quinones are known

reactive Michael acceptors, they were categorized under

this electrophilic reaction mechanism domain. Structure-

activity trends were evaluated within this mechanism

domain as well as by structural similarity. The variation

between each derivative is limited to the substituents

present on the 2,3,5 and 6 carbons of the aromatic ring

template (Table 1). Rat hepatocyte cytotoxicity for this

group of p-benzoquinones correlated best to the energy of

the lowest unoccupied molecular orbital (ELUMO) (Fig. 1).

Outliers were determined as data points that had residuals

exceeding three sigma (Hall et al., 2003; Huuskonen,

2001; Lipnick, 1991). Removal of 2-tert butyl benzoquin-

one as an outlier resulted in the derived parabolic equation:

log LC50 = 24.54 + 17.7 ELUMO + 3.36 (ELUMO)2;

n = 9, r2 = 0.90, P = 0.002 (1)

ELUMO serves as a global reactivity parameter which

measures the readiness of a molecule to accept electrons,

i.e. electrophilicity. The derived parabolic line of best fit

suggests that at first cytotoxicity increases as the values

of ELUMO become more negative, but then at a specific

point cytotoxicity turns around and decreases. An ELUMO

value of −2.63 eV is where the inversion point occurs,

which theoretically is the most cytotoxic p-benzoquinone.

Table 4 describes the electron density at each carbon

atom on the aromatic ring. LUMO energy and nucleo-

philic frontier density (NuFD) at each carbon atom was

calculated. Electron density on C′2 (see Table 1 for

carbon positions) described by LUMO and NuFD corre-

lated best with rat hepatocyte cytotoxicity (Fig. 2a,b). The

following QSAR models were derived:

log LC50 = 11.02 − 91.92 C′2 LUMO;

n = 9, r2 = 0.79, P = 0.001 (2)

log LC50 = 10.34 − 42.35 C′2 NuFD;

n = 9, r2 = 0.77, P = 0.002 (3)

Chloranil was an outlier present in both models. Both

LUMO and NuFD at C′2 atom describe the same



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Table 4. Electron density properties for carbon atoms on the aromatic ring

LUMO density (LUMO) Nucleophilic frontier density (NuFD)

p-Benzoquinone C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C6

1 chloroanil 0.147 0.098 0.098 0.147 0.098 0.098 0.291 0.197 0.197 0.291 0.197 0.197

2 2,5-dichloro-benzoquinone 0.154 0.106 0.092 0.154 0.106 0.092 0.306 0.213 0.184 0.306 0.213 0.183

3 2-bromo-benzoquinone 0.155 0.104 0.108 0.16 0.094 0.094 0.308 0.212 0.214 0.319 0.186 0.1884 2-tert butyl-benzoquinone 0.166 0.102 0.087 0.169 0.102 0.105 0.332 0.204 0.173 0.337 0.203 0.21

5 2-methyl-benzoquinone 0.172 0.099 0.087 0.156 0.103 0.096 0.345 0.199 0.174 0.313 0.207 0.193

6 p-benzoquinone 0.163 0.098 0.098 0.163 0.098 0.098 0.326 0.196 0.196 0.326 0.196 0.1967 2,6-dimethyl-benzoquinone 0.181 0.097 0.092 0.151 0.092 0.097 0.362 0.195 0.184 0.301 0.184 0.195

8 2,6-dimethoxy-benzoquinone 0.202 0.103 0.077 0.124 0.077 0.103 0.403 0.207 0.153 0.246 0.153 0.207

9 2,3,5-trimethyl-benzoquinone 0.16 0.093 0.086 0.175 0.104 0.091 0.319 0.186 0.172 0.349 0.208 0.18210 2,3,5,6-tetramethyl-benzoquinone 0.169 0.092 0.092 0.169 0.092 0.092 0.337 0.184 0.184 0.337 0.184 0.184

electronic property, which is electron density based on

the distribution of electrons in or near the lowest unoccu-

pied electron orbital. The electron density properties of

the other carbon atoms did not derive any relationship

with cytotoxicity in rat hepatocytes.

Human Hepatocytes

MLR analysis for p-benzoquinone induced cytotoxicity in

human hepatocytes did not show any significant correla-

tion to the structural parameters studied. The parameters

that showed some weak sign of correlations were ELUMO

and molecular refractivity (MR), both of which gave

parabolic equations with r2 values of 0.55 and 0.54

respectively (Table 5; eq. 4 and 5). However, both re-

sulted in P values of 0.1, indicating that the correlation

is not significant. Analysis of the electron density at each

carbon atom resulted in correlation between LUMO density

and NuFD at C′1 of the benzoquinone with cytotoxicity

in human hepatocytes. The exclusions of 2-Br and

Figure 2a. Plot of log LC50 cytotoxicity values for substituted p-benzoquinones in rat hepatocytes against LUMOdensity at C′2. The line represents Eqn. (2): log LC50 = 11.02 − 91.92 C′2 LUMO, n = 9, r2 = 0.79, s = 0.25, F = 25.5,P = 0.001 (�) correlated data; (�) outlier

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log LC50 = 3.22 − 3.21 C′1 NuFD;

n = 8, r2 = 0.57, P = 0.03 (7)

Both correlations were of moderate strength, both having

r2 values of approximately 0.55.

2,3,5,6-(CH3)4 benzoquinones as outliers were required to

derive the following QSAR equations (Fig. 3a,b):

log LC50 = 3.24 − 6.5 C′1 LUMO;

n = 8, r2 = 0.56, P = 0.032 (6)

Figure 2b. Plot of log LC50 cytotoxicity values for substituted p-benzoquinones in rat hepatocytes against NuFD atC′2. The line represents Eqn. (3): log LC50 = 10.34 − 42.35 C′2 NuFD, n = 9, r2 = 0.77, s = 0.23, F = 24, P = 0.002, (�)correlated data; (�) outlier

Table 5. QSAR models derived for p-benzoquinone toxicity and reactivity

Equation n r2 s F P outlier

Rat hepatocyte cytotoxicity

(1) log LC50 = 24.54 + 17.7 ELUMO + 3.36 (ELUMO)2 9 0.90 0.04 25.9 0.002 -2(CH3)4

(2) log LC50 = 11.02 − 91.92 C′2 LUMO 9 0.79 0.25 25.5 0.001 -Cl4

(3) log LC50 = 10.34 − 42.35 C′2 NuFD 9 0.77 0.23 24.0 0.002 -Cl4

Human hepatocyte cytotoxicity

(4) log LC50 = 28.29 + 21.65 ELUMO + 4.42 (ELUMO)2 9 0.55 0.08 3.7 0.100 2Br

(5) log LC50 = 7.59 − 0.32 MR + 0.004 MR2 9 0.588 0.09 3.6 0.100 2Br

(6) log LC50 = 3.24 − 6.5 C′1 LUMO 8 0.56 0.11 7.8 0.032 2Br, 2,3,5,6-(CH3)4

(7) log LC50 = 3.22 − 3.21 C′1 NuFD 8 0.57 0.11 7.97 0.03 2Br, 2,3,5,6-(CH3)4

GSH thiol reactivity

(8) log kGSH = −18.38 − 16.78 ELUMO − 3.19 (ELUMO)2 10 0.80 0.07 13.79 0.008 None

GSH thiol reactivity and cytotoxicity

(9) log LC50 (rat) = 4.65 − 0.92 log kGSH 10 0.82 0.23 35.24 <0.001 None

(10) log LC50 (human) = 3.76 − 0.47 log kGSH 10 0.40 0.32 5.1 0.05 None



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Figure 3a. Plot of log LC50 cytotoxicity values for substituted p-benzoquinones in human hepatocytes againstC′1 LUMO density. The line represents Eqn. (6) log LC50 = 3.24 − 6.5 C′1 LUMO; n = 8, r2 = 0.56, s = 0.11, F = 7.8,P = 0.032. (�) correlated data; Outliers not shown

GSH Thiol Reactivity

Quinones are well known electrophiles which can alkylate

glutathione and protein thiols in the cell. Non-enzymic

thiol reactivity of benzoquinones based on glutathione

depletion for the measurement of soft electrophilic reac-

tivity was measured as kGSH (M−1 min−1) (Table 2).

The most reactive benzoquinones were 2,5-Cl2 and

chloranil, which depleted glutathione rapidly. The least

reactive was 2,3,5,6-(CH3)4. 2,3,5,6-(CH3)4 was very

poorly reactive with GSH as almost no conjugation

of glutathione was detected in the assay, even when the

incubation time was increased (data not shown). The

order of reactivity from fastest to slowest was: 2,5-Cl2

> Cl4 > 2CH3 > BQ > 2,6-(CH3)2 > 2-Br > 2-tbutyl > 2,6-

(OCH3)2 > 2,3,5-(CH3)3 > 2,3,5,6-(CH3)4.

QSAR and GSH Thiol Reactivity

MLR analysis revealed that the non-enyzmic rate of GSH

conjugation by p-benzoquinones depended also on ELUMO.

The derived QSAR model (Fig. 4) was parabolic and is

represented by the following equation:

log kGSH = −18.38 − 16.78 ELUMO − 3.19 (ELUMO)2;

n = 10, r2 = 0.80, P = 0.008 (8)

No outliers were present in this derivation. The parabolic

line of best fit suggests that GSH reactivity increases

as ELUMO values become more negative but then at a

specific point, approximately −2.62 eV, GSH reactivity

turns around and decreases. This trend is similar to the

relationship derived for hepatocyte toxicity. Thus p-

benzoquinones with low ELUMO values up to the inversion

point are not only more cytotoxic but also are more GSH

reactive.

GSH Thiol Reactivity and Cytotoxicity

Liver toxicity is an acute endpoint measure for toxi-

city. While glutathione reactivity is a surrogate model

that represents the activity of covalent binding

which is a central unifying event for many toxicity path-

ways. In analysing rat hepatocyte cytotoxicity (LC50)

and its relationship with GSH reactivity (kGSH) for p-

benzoquinones, the following correlation was derived

(Fig. 5):

616 K. CHAN ET AL.


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Figure 3b. Plot of log LC50 cytotoxicity values for substituted p-benzoquinones in human hepatocytes against C′1NuFD. The line represents Eqn. (7) log LC50 = 3.22 − 3.21 C′1 NuFD; n = 8, r2 = 0.57, s = 0.11, F = 7.97, P = 0.03. (�)correlated data; Outliers not shown

Log LC50 = 4.65 − 0.92 log kGSH;

n = 10, r2 = 0.82 and P < 0.001 (9)

No outliers were apparent in this model. This indicates

that there is a strong and significant correlation between

the reactivity of benzoquinones with nucleophiles and rat

hepatocyte toxicity. Comparison of GSH reactivity and

cytotoxicity in human hepatocytes resulted in a much

weaker relationship compared with that found in the rat

model:

log LC50 = 3.76 − 0.47 log kGSH;

n = 10, r2 = 0.40, P = 0.05 (10)

Discussion

The hepatotoxic potential and electrophilic reactivity

of ten substituted p-benzoquinones were investigated

here. QSAR modeling was also explored to assess the

physico-chemical parameters which underlie quinone

hepatotoxicity in relation to their electrophilic reactivity

with glutathione.

In rat hepatocytes the most cytotoxic benzoquinone

was chloranil, a tetra-chloro substituted benzoquinone,

while the least cytotoxic was the tetra methylated

benzoquinone, 2,3,5,6-(CH3)4. In human hepatocytes, 2,6-

dimethyl benzoquinone was the most toxic, and similar

to rat hepatocytes duroquinone was the least toxic. To

understand the reason for the higher toxicity of the

dimethyl benzoquinone in human hepatocytes versus

chloranil in rat hepatocytes, a more detailed investigation

would be needed to compare the effects of metabolizing

and detoxifying enzymes in the hepatocytes of these two

species.

Frontier molecular orbital energies have been shown to

play a major role in governing many chemical reactions.

They are responsible for the formation of charge transfer

complexes involved in chemical reactions and have

become commonly used quantum chemical descriptors

for molecular interactions. The energy (E) of the lowest

unoccupied molecular orbital (LUMO) serves as a global

reactivity parameter which measures the energy added

when an electron is placed in the LUMO to produce

a negative ion (electron affinity). ELUMO essentially char-

acterizes the susceptibility of the molecule toward attack



DOI: 10.1002/jat

Figure 4. Plot of log kGSH reactivity versus ELUMO values for substituted p-benzoquinones. The line represents Eqn.(8): log kGSH = −18.38 − 16.78 ELUMO − 3.19 (ELUMO)2; n = 10, r2 = 0.80, s = 0.07, F = 13.79, P = 0.008. (�) correlated data

by nucleophiles (Karelson et al., 1996). The calculated

ELUMOs for the p-benzoquinones studied were low within

the range of −3.00 to −1.00, which is typical for soft elec-

trophilicity. The QSAR model derived for the cytotoxicity

in rat hepatocytes suggests that the p-benzoquinones with

lower more negative LUMO energies are more cytotoxic

than higher energy LUMO compounds. However, after a

certain point, cytotoxicity turns around and decreases with

lower ELUMO values. This may be attributed to a change in

the mechanism for compounds with larger negative ELUMO

values. Chloranil which had the lowest ELUMO, −2.832,

in the group was the most cytotoxic (LC50 = 18 μM).

2,3,5,6-(CH3)4 which was the least toxic benzoquinone,

44 times less toxic than chloranil, and had the highest

ELUMO of −1.976. 2-Tertbutyl was an outlier in this deriva-

tion. The tert butyl group likely sterically hinders Michael

type addition reactions on the ring making it an outlier in

this group and acting by a different mechanism.

The different substituents on the ten p-benzoquinones

studied ranged from being strongly electron withdrawing

to electron donating, covering a diverse range of electronic

properties. The electronic effect of substituents on the

intrinsic reactivity and cytotoxicity of p-benzoquinones

was explored. The results show that the electron donating

or electron withdrawing ability of the substituent on the

benzoquinone has a role in directing the pattern of cyto-

toxicity in rat hepatocytes. The less cytotoxic benzoquin-

ones of this series were substituted with electron donating

groups. The electron donating substituent varied by their

positions and number of methyl groups on the aromatic

ring. Methyl groups by their electron-donating effects

make quinones less electrophilic depending on their

position relative to the reactive center. Thus, it is not

surprising that the order of toxicity is CH3 > 2,6-(CH3)2

> 2,3,5-(CH3)3 > 2,3,5,6-(CH3)4, and this order correlated

to a global increase in electrophilicity, as described by

the low ELUMO.

The p-benzoquinones substituted with electron withdraw-

ing groups included –Cl, –Br and –OCH3 substituents. In

contrast to electron donating groups, electron withdraw-

ing groups remove density from the pi system of the aro-

matic ring, making it more electrophilic. Br substitution

was less toxic than Cl substitution; which is in accord

with the measure of electronegativity of the halogen. The

more electronegative substituent, Cl, is more strongly

electron withdrawing; therefore it was better at activating

the aromatic ring.

Since substituents influence chemical reactivity, the

atomic properties for each carbon position on the benzo-

quinone ring were considered. The electron density at

618 K. CHAN ET AL.


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Figure 5. Plot of p-benzoquinone rat hepatocyte cytotoxicity against GSH thiol reactivity. The correlation is repre-sented by the Eqn (9): log LC50 = 4.65 − 0.92 log kGSH, n = 10, r2 = 0.82, s = 0.23, F = 35.24, P < 0.001 (�) correlated data

each carbon was investigated and QSAR models were

derived for its effect on cytotoxicity. NuFD and LUMO

density were calculated for each of the six carbon centers

on the benzoquinone structure. The frontier orbital

electron density studied here, NuFD and LUMO, meas-

ures the relative reactivity of the lowest unoccupied

molecular orbital for a specific atom within the molecule.

This is different from ELUMO discussed previously, which

reflects the relative reactivity of molecules globally.

Figures 2a,b show that cytotoxicity towards rat hepato-

cytes correlated to higher values of NuFD and LUMO

density at C′2 position. C′2 is part of the double bond

which is subject to nucleophilic attack and in the α-position

relative to the carbonyl carbon (C′1). The increase in

electron density at this carbon position influences toxi-

city, as it withdraws electron density from the electro-

philic center stabilizing the carbocation for nucleophilic

addition of the thiol. Chloranil was an outlier in these

atomic QSAR models. Its presence as an outlier can be

explained by its different mechanism of action. Chloranil

is fully substituted with four Cl atoms, because of this it

reacts with nucleophiles by direct substitution followed

by the loss of HCl rather than by reductive addition seen

by Michael type reactions (O’Brien, 1991).

Overall, the results suggest that electrophilicity meas-

ured by ELUMO, which measures electron affinity and the

ease of reduction, is a good measure of potential toxicity

induced by quinones. Consistent with these findings,

O’Brien (1991) previously suggested that quinones

with higher redox potential are strongly electronegative

and therefore are more cytotoxic and reactive towards

GSH conjugation. Typically Hammett substituent con-

stants are used in QSAR studies to quantify the electronic

effect of substituents on reaction rates (Bathelt et al.,

2004) and interpret electrophilicity. However, Hammett

parameters are only available for a limited number of

substituents due to the experimental effort involved.

Molecular orbital energies such as ELUMO for determining

intrinsic electronic properties can be calculated semi-

empirically using a computer and therefore should be

considered as a convenient alternative for measuring

electrophilicity.

Non-enzymatic GSH reactivity also correlated best

with ELUMO (Fig. 4, Table 5, Eqn. 8). This model suggests

that thiol reactivity increased with electrophilicity. The

p-benzoquinones with lower ELUMO more readily accept

electrons to form a covalent bond with the thiol group of

GSH. No outliers were present in this QSAR model.



DOI: 10.1002/jat

Quinones react with GSH via the 1,4-Michael addition

mechanism, producing GSH hydroquinone conjugates

in vitro (O’Brien, 1991). Substitution with electron donat-

ing substituents like, –CH3, and –OCH3, decreased the

rate of GSH addition. While those with electron with-

drawing substituents, –Br or –Cl, increased reactivity.

To confirm that measuring GSH reactivity can reflect the

mechanistic reactivity of quinones to cause cytotoxicity

by alkylation, a relationship between GSH reactivity

and rat hepatotoxicity was assessed. A strong and signifi-

cant correlation between LC50 and kGSH (Fig. 5, Table 5,

Eqn. 9) was derived. This verified that GSH reactivity is

a good indicator of p-benzoquinone cytotoxicity in rat

hepatocytes and both are dependent on the electrophilicity

of the p-benzoquinone.

Similar to this study, Siraki et al. (2004) showed that

electron affinity (EA) was the best parameter for describing

p-benzoquinone induced cytotoxicity and GSH depletion

in rat hepatocytes. EA is essentially the positive value of

ELUMO. Siraki suggested that EA parameterizes for one

electron reduction pathways that leads to ROS formation,

presumably from redox cycling and also it may partially

parameterize alkylation pathways, however, another variable

may be required. In this study, the results confirm that

ELUMO parameterizes for an alkylation pathway.

In human hepatocytes cytotoxicity was moderately

correlated to ELUMO and molecular refractivity (MR;

describes the overall molecular bulk based on size and

polarizability), however, the correlation failed at the

statistical validation stage, P = 0.1. Cytotoxicity also

moderately correlated to electron density described by

NuFD and LUMO at C′1. C′1 is part of the carbonyl

moiety, which has electron withdrawing ability, thus

lowering the electron density in the ring increasing the

likelihood for nucleophilic conjugation of thiol groups.

The relationships derived in Eqns 6 and 7, suggests that

the higher the energy of the unoccupied orbital localized

on the double bond of this carbon, the more toxic the

compound. Increases in the energy of the lowest unoccu-

pied orbital increases the electron density on this carbon.

This affects the electrostatic and polarization component

of the molecule at the electrophilic center, increasing its

susceptibly to thiol addition (Osman et al., 1988). No

robust relationships were found in any of these models.

The structure-activity conclusion that can be drawn as yet

is that sterics in addition to electrophilicity has an impor-

tant role in the cytotoxic mechanism of p-benzoquinones

towards human hepatocytes. Non-enzymic GSH reactiv-

ity and human hepatocyte toxicity also had a relatively

weak relationship in comparison with what was found for

rat hepatocytes. Unsuccessfully, no relationship was con-

firmed between human and rat hepatocyte cytotoxicity.

The differences in strength of the human models com-

pared with the rat may be due partly to differences in the

preparation of the hepatocytes. Thawing of cryopreserved

hepatocytes itself can result in a 10–20% loss in viabil-

ity and yield. This loss is minimal and considered accept-

able because of the relatively high viability of the re-

maining cells (Li et al., 1999) and is therefore more or

less on a par with fresh hepatocytes. Sohlenius-Sternbeck

and Schmidt (2005), demonstrated that cryopreserved

hepatocytes from rat and humans had much lower levels

of intracellular glutathione than freshly prepared rat

hepatocytes (approx 18-fold less) and suggests that the

cryopreservation procedure decreased the hepatocyte

glutathione conjugating capacity (Sohlenius-Sternbeck

and Schmidt, 2005). This may explain the less robust

viability derived for human hepatocytes and the poor

correlation to non-enzymic GSH reactivity. Nevertheless,

the derived models although statistically poorer for human

hepatocytes, point in the direction of electrophilic reactiv-

ity as an important indicator for potential liver toxicity.

In this study correlation of p-benzoquinone induced rat

and human hepatocyte cytotoxicity with other parameters

such as log P, EHOMO, redox potential, dipole moment and

molecular refractivity were not significant compared with

ELUMO or the atomic frontier electron density descriptors

on C′2 and C′1. Normally in toxicity QSAR modeling,

increases in hydrophobic character results in increased

toxic potency. However, in this study hepatocyte

cytotoxicity for all ten analogs did not correlate to log P.

Generally log P is related to biomembrane permeation

of the toxicant. However, in addition to uptake, log P in

mammalian cells has been related to intracellular meta-

bolism which includes binding, induction and inhibition

of phase I and II metabolizing enzymes (Chan et al.,

2007). The insignificance of log P in this study suggests

that membrane permeation and interaction with quinone

metabolizing enzymes, particularly, membrane bound

reductases are not rate limiting steps in the toxicity

mechanism, and that electrophilic reactivity has a more

significant role.

There is an increasing need to be able to predict toxic

potency for risk assessment and regulatory purposes for

both environmental and human endpoints (Schultz et al.,

2007). It is also necessary in drug development to reduce

the number of unnecessary toxicities that are seen in drugs

released on to the market (Guengerich and Macdonald,

2007). Protein covalent binding by reactive drug meta-

bolites is recognized to be a critical event in initiating

organ toxicities such as hepatotoxicity (Guengerich and

Macdonald, 2007). Application of QSAR to this process

has concentrated on describing molecular triggering

events in toxicity such as protein covalent binding, rather

than on typical toxicity endpoints which are complex.

It has been shown that for substituted p-benzoquinones

their rate of soft electrophilic reactivity with GSH can

potentially predict cytotoxicity in rat hepatocytes. As a

first step, the potential for surrogate models of a critical

event in toxicity to improve toxicity-based QSAR

models was shown. QSAR is a tool that should be looked

upon as an aid in chemical risk assessments, and an

620 K. CHAN ET AL.


DOI: 10.1002/jat

alternative non-animal method for predicting toxicity

that is easy and efficient in terms of time and financial

costs (Lessigiarska et al., 2006). However, as advised by

Harder et al. (2003) the potential influence of metabolism

on the occurrence of reactive modes of toxic action

should be realized, and therefore we should be looking

for toxicity indicators that clearly link observable toxi-

city with mechanisms of toxic action. QSARs should not

be looked at as a final decision predictive tool, but rather

as an exploratory tool to provide insight into the func-

tionalities of structure on a particular biological effect

(Harder et al., 2003). This method may help to screen out

potentially toxic compounds during the early drug discov-

ery stages and help access risks for chemical toxins.

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