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Toxicology 290 (2011) 295–304

Contents lists available at SciVerse ScienceDirect

Toxicology

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onic mechanisms underlying cardiac toxicity of the organochloride solventrichloromethane

uan Zhoua,b, Hui-Jun Wub, Yan-Hui Zhanga, Hai-Ying Sunb, Tak-Ming Wonga, Gui-Rong Lia,b,∗

Department of Physiology, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Pokfulam, Hong Kong, ChinaDepartment of Medicine, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Pokfulam, Hong Kong, China

r t i c l e i n f o

rticle history:eceived 27 August 2011eceived in revised form 12 October 2011ccepted 12 October 2011vailable online 17 October 2011

eywords:richloromethaneardiac toxicityrrhythmogenic effectentricular fibrillation

a b s t r a c t

Trichloromethane (chloroform) is widely used for industrial chemical synthesis and also as an organicsolvent in laboratories or ingredient of pesticides. Sudden death resulted from cardiac arrhythmias hasbeen reported in clinic with acute trichloromethane intoxication. The present study was designed toinvestigate ionic mechanisms underlying arrhythmogenic effect (cardiac toxicity) of trichloromethanein isolated rat hearts and ventricular myocytes and HEK 293 cells stably expressing human Nav1.5, HCN2,or hERG channel using conventional electrophysiological approaches. It was found that trichloromethane(5 mM) induced bradycardia and atrial-ventricular conduction blockade or ventricular fibrillation, andinhibited cardiac contractile function in isolated rat hearts. It shortened action potential duration (APD) inisolated rat ventricular myocytes, and increased the threshold current for triggering action potential, buthad no effect on the inward rectifier K+ current IK1. However, trichloromethane significantly inhibited the

ultiple ion channel blockade L-type calcium current ICa.L and the transient outward potassium current Ito in a concentration-dependentmanner (IC50s: 1.01 and 2.4 mM, respectively). In HEK 293 cells stably expressing cardiac ion channelgenes, trichloromethane reduced hNav1.5, HCN2, and hERG currents with IC50s of 8.2, 3.3, and 4.0 mM,respectively. These results demonstrate for the first time that trichloromethane can induce bradycardiaor ventricular fibrillation, and the arrhythmogenic effect of trichloromethane is related to the inhibition

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of multiple ionic currents

. Introduction

Trichloromethane (chloroform) was used as an inhaling anes-hetic for general anesthesia in 19th century, and then it waserminated in clinic usage due to its toxicity and side effects,ncluding cardiac arrhythmias, headache, dizziness, convulsions,espiratory paralysis and disturbance of autonomic nervous sys-em (Payne, 1981; Schroeder, 1965; Whitaker and Jones, 1965).owever, this organochlorine solvent is still widely used for indus-

rial synthesis of fluorocarbons and tetrafluoroethylenes and alsos organic solvent in laboratories and pesticides production. Lethalardiac arrhythmias have been reported after intoxication withrichloromethane in cases of suicide and homicide (Meichsner etl., 1998; Nadjem and Logemann, 1998; Risse et al., 2001). Therichloromethane-induced cardiac arrhythmia (toxicity) reported

n clinical patients was believed to be related to sensitization ofhe heart to catecholamine (Levy, 1911; Muller et al., 1997). Simi-ar effects were found in a group of clinical volatile anesthetic and

∗ Corresponding author at: L4-59, Laboratory Block, FMB, the University of Hongong, 21 Sassoon Road, Pokfulam, Hong Kong, China. Tel.: +852 2819 9513;

ax: +852 2816 2095.E-mail address: grli@hku.hk (G.-R. Li).

300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2011.10.009

ding ICa.L, Ito, INa, HCN2, and hERG channels.© 2011 Elsevier Ireland Ltd. All rights reserved.

halogenated hydrocarbons, such as halothane, trichloroethylene,etc. (Brock et al., 2003; Hayashi et al., 1991; Himmel, 2008; Lessardet al., 1977; Vinegar, 2001). Recently, Jiao and colleagues reportedthat the cardiac sensitization to catecholamine might be due to theslowed conduction caused by reduced phosphorylation of connexin43 resulting in gap junction uncoupling (Jiao et al., 2006).

Study of direct effect of trichloromethane on cardiacion channels is scarce. A report recently demonstrated thattrichloromethane inhibited hERG channel (Scholz et al., 2006).However, the effects of trichloromethane on other cardiac ionchannels are unknown. The present study was therefore designedto investigate the ionic mechanisms underlying arrhythmogeniceffect of trichloromethane in isolated rat hearts and ventricularmyocytes, and HEK 293 cell lines expressing human cardiac ionchannels. We found that trichloromethane cardiac toxicity wasrelated to inhibition of multiple ionic currents, including L-typeCa2+ current (ICa.L), transient outward K+ current (Ito), voltage-gated sodium current (INa), HCN2 current, and hERG current, butnot the inward rectifier K+ current IK1.

2. Material and methods

2.1. Animals

Adult male Sprague–Dawley (SD) rats weighing 250–350 g were provided by theLaboratory Animal Unit of University of Hong Kong. The animals were cared with

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he guide for the Care and Use of Laboratory Animals as adopted and promulgatedy the United States National Institutes of Health. The experimental protocol waspproved by the Committee of Animal Use for Teaching and Research, University ofong Kong.

.2. Reagents and solutions

Collagenase type II was purchased from Worthington Biotech (NJ, USA). Bovineerum albumin and protease type XXIV were purchased from Sigma–Aldrich (St.ouis, MO). All other chemicals and reagents were purchased from Sigma–Aldrich.

Kreb’s solution containing (in mM) NaCl 118.0, KCl 5.0, CaCl2 1.25, KH2PO4 1.2,gSO4 1.2, NaHCO3 25.0, and glucose 11.0 (pH 7.2) was used for Langendorff perfu-

ion during ECG and cardiac contractile function recording in the isolated rat hearts37 ◦C). Tyrode solution contained (in mM): NaCl 140, KCl 5.0, MgCl2 1.0, CaCl2 1.8,-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10.0, NaH2PO4 0.33nd glucose 10 (pH adjusted to 7.3 with NaOH). CdCl2 (200 �M) was added to theyrode solution for IK1 recording, and both BaCl2 (200 �M) and CdCl2 were addedor Ito recording.

Kraftbrühe (KB) solution containing (in mM) 70 potassium glutamate, 25 KCl,0 taurine, 10 KH2PO4, 3 MgCl2, 0.5 EGTA, 10 glucose, and 10 HEPES-KOH (pH.2) was used for preserving isolated cardiac myocytes. The pipette solution (for+ current recording) contained (in mM): 110 K-aspartate, 20 KCl, 1 MgCl2, 5 Na-hosphocreatine, 10 HEPES, 0.05 K2-EGTA, 0.1 GTP, and 5 Mg-ATP. The pH wasdjusted to 7.2 with KOH. For INa and ICa.L recordings, K+ was replaced by equimolars+, and the pH was adjusted to 7.2 with CsOH.

.3. Isolated heart preparation and ECG recording

Male Sprague–Dawley rats were sacrificed by cervical dislocation after intraperi-oneal injection of sodium pentobarbital (60 mg/kg, i.p.). Their hearts were isolatednd retrogradely perfused on a Langendorff perfusion apparatus with a constantressure (80 cm H2O) at 37 ◦C with Kreb’s solution equilibrated with 95% O2/5% CO2.olyethylene tubing with a cling film balloon tip filled with water and mounted onpressure transducer was inserted into the left ventricle through a small incision in

he left atrium to measure left ventricular pressure. Electrodes were placed on theeart surface to record electrocardiogram (ECG) (Cao et al., 2005; Liu et al., 2010). Theiological signals were recorded using a PowerLab System (ML750 PowerLab/4sp;D Instruments, Colorado Springs, CO). Heart rate was measured using the RR inter-al. PR interval was measured from the beginning of P wave to the beginning of QRSomplex. QT interval was measured from the beginning of QRS complex to the end ofwave. QTc (heart rate-corrected QT interval) was calculated with Van de Water’s

ormula (Van de Water et al., 1989): QTc = QT-0.087 × (RR-1000), where the unitf the RR interval is milliseconds. The result exhibited a good correlation with theepolarization period of the heart (Takahara et al., 2005).

.4. Isolated ventricular myocytes

Ventricular myocytes were isolated from the heart of SD rats after anaesthetiza-ion with sodium pentobarbital (60 mg/kg, i.p.) as described previously (Gao et al.,004). Briefly the Langendorff heart was perfused with normal Tyrode solution formin to wash out the blood in the heart. The heart was then further perfusedith Ca2+-free Tyrode solution for 5 to 10 min, followed by Ca2+-free Tyrode solu-

ion containing 0.3 mg/mL collagenase (type II, Worthington), 0.2 mg/mL proteasetype XXIV, Sigma–Aldrich) and 1 mg/mL bovine serum albumin (Sigma–Aldrich)or 20–30 min. All the perfusing solutions were bubbled with 100% oxygen and

aintained at 37 ◦C. After the digestion, ventricles were removed from the soft-ned hearts and stored in KB solution. Single myocytes were suspended by gentleipetting and were kept in the KB solution at room temperature for 2 h before patchlamp experiments.

.5. Cell culture

Human embryonic kidney (HEK) 293 cell lines stably expressed hERG/pcDNA3generously provided by Dr. G. Robertson, University of Wisconsin, WI, USA), humanir2.1/pcDNA3 (generously provided by Dr. Carol A. Vandenberg, University of Cal-

fornia, CA, USA), human SCNA5/pcDNA3 (i.e. Nav1.5, kindly provided by Dr. J.akielski, Wisconsin University, USA), human HCN2/pcDNA3 (kindly provided byr. A. Ludwig, Friedrich Alexander Universität Erlangen Nürnberg, Germany) (Tangt al., 2008; Zhang et al., 2008, 2009) were cultured in Dulbecco’s modified Eagleedium (DMEM; Invitrogen, Carlsbad, CA). The culture medium contained 10% fetal

ovine serum with 400 �g/ml G418 for selection and incubated in 5% CO2 and 95%ir at 37 ◦C. The cells were seeded on a glass cover slip overnight for the electro-hysiological recording.

.6. Electrophysiology

One drop of KB aliquot solution containing the isolated myocytes was placedn an open chamber (1 mL) mounted on the stage of an inverted microscope forlectrophysiological recording. The cells were allowed to settle to the bottom ofhe chamber for 5–10 min and superfused with Tyrode solution at ∼2 mL/min. Only

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quiescent rod-shaped myocytes with clear cross-striations were used for experi-ments. For HEK 239 cell lines, cells seeded on a cover slip were transferred to anopen chamber and then superfused with Tyrode solution for the current recordings.

Action potential and membrane current were recorded by whole-cell patchclamp technique in current clamp mode and voltage-clamp mode, respectively,using an EPC-10 amplifier and Pulse software (Heka Elektronik, Lambrecht,Germany). A 3-M KCl agar bridge was used as the reference electrode. Borosili-cate glass electrode pipettes (1.2 mm OD) were pulled by Brown-Flaming puller(Model No. P-97, Sutter Instrument Co, Novato, CA, USA), and had tip resistancesof 1.5–2.5 M� when filled with pipette solutions. Tip potentials were compensatedbefore the patch pipette contacted the cell. After a giga-ohm seal was obtained, thecell membrane was ruptured by gentle suction to establish whole-cell configura-tion. Series resistance (Rs) was compensated by 60–80% to minimize voltage errors.Cell membrane capacitive transient was compensated electronically. Liquid junc-tion potentials (11–15 mV) between pipette and bath solutions were not adjustedduring current or voltage clamp experiments. Current and/or voltage signals werelow-pass filtered at 5 kHz and stored in the hard disk of a PC computer for offlinedata analysis.

2.7. Data analysis

All results were expressed as means ± SEM. Nonlinear curve fitting was per-formed using Pulsefit (HEKA, Lambrecht/Pfalz, Germany) and Sigmaplot 8.0 (SPSS,Chicago, Ill, USA). Paired and/or unpaired Student’s t-test was used for two groups.Multiple group data were compared using analysis of variance, then with a Dunnettpost hoc test to evaluate the statistical significance of the differences between groupmeans. A value of P < 0.05 was considered statistical significant.

3. Results

3.1. Effects of trichloromethane on electrical activity andcontractile function in isolated heart

The potential effects of trichloromethane on cardiac electri-cal activity and contractile function were determined in isolatedrat hearts. Fig. 1 shows the recording traces of ECG and leftventricular development pressure (LVDP) and the related param-eters in isolated rat hearts in the absence and presence of 5 mMtrichloromethane. In a total of 12 experiments, seven hearts exhib-ited bradycardia and/or atrial-ventricular conduction blockade(58%) and reduction of LVDP; the effect was reversible on washout(Fig. 1A). Other five hearts (42%) showed an initial bradycardiaand subsequently ventricular tachycardia/fibrillation. This effectwas not reversed by washout (Fig. 1B). The ECG parameters areillustrated in Fig. 1C before and after 5 mM trichloromethaneadministration for 5 min. Trichloromethane slowed heart rate, pro-longed PR interval in isolated rat hearts (n = 7, P < 0.01 vs. control),and slightly increased QRS complex. In hearts without ventriculartachycardia/fibrillation, LVDP and contraction/relaxation velocity(±dp/dtmax, mmHg/s) (Fig. 1D) were significantly reduced by 5 mMtrichloromethane (P < 0.01 vs. control).

3.2. Effect of trichloromethane on action potentials in ventricularmyocytes

To explore the potential cellular mechanisms underlying cardiacarrhythmias, the effect of trichloromethane on action potentialswas studied in isolated ventricular myocytes with whole-cell patchclamp in current clamp mode. Fig. 2A displays the action poten-tials recorded in a representative myocyte before and after 5 mMtrichloromethane for 5 min. Trichloromethane markedly short-ened the action potential duration (APD), and induced a triangleshaped action potential, suggesting a reduced ICa.L. Changes inaction potential parameters with 5 mM trichloromethane are illus-trated in Fig. 2C. APD20 (time to 20% repolarization), APD50, andAPD90 were significantly reduced. In addition, the current thresh-

old for triggering action potential was significantly increased(Fig. 2B) from 733 ± 19 pA in control to 1117 ± 148 pA with 5 mMtrichloromethane (n = 8, P < 0.05 vs control). No change in the rest-ing membrane potential was observed.

Y. Zhou et al. / Toxicology 290 (2011) 295–304 297

Fig. 1. Effect of trichloromethane (chloroform) in isolated rat hearts. A. LVDP and ECG traces recorded in a representative isolated rat heart during control, after perfusing5 mM trichloromethane (5 min) and washout (10 min). B. LVDP and ECG traces recorded in another isolated rat heart during control, after perfusing 5 mM trichloromethanef ts (n =d (contfi

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or 5 min and washout (10 min). C. ECG parameters recorded in isolated rat hearevelopment pressure; ±dP/dtmax, systolic and diastolic velocity) recorded beforebrillation (n = 7, **P < 0.01 vs. control).

.3. Effect of trichloromethane on IK1

Fig. 3A displays the IK1 traces recorded with the typical hyper-olarization voltage protocol (Li et al., 2004) as shown in the inset

n a representative ventricular myocyte in the absence and pres-nce of 10 mM trichloromethane. Trichloromethane had no effectn IK1 amplitude. Fig. 3B illustrates the current–voltage (I–V) rela-ionships of IK1 before and after application of trichloromethane.o significant change was observed with 10 mM trichloromethanet test potentials of −120 to −20 mV. Similar results were found inEK cell line expressing human Kir2.1 gene (n = 6, data not shown).hese results are consistent with observations of resting membraneotential in current clamp mode.

.4. Inhibition of ICa.L by trichloromethane

Fig. 4A shows the voltage-dependent ICa,L traces recorded inrepresentative cell using the voltage protocol as showed in the

7, *P < 0.05 vs. control). D. Cardiac contractile parameters (LVDP, left ventricularrol) and after trichloromethane application (5 min) in hearts without ventricular

inset. ICa,L was remarkably inhibited by 10 mM trichloromethane(5 min), and the effect was partially reversed by washout (10 min).I–V relationships of ICa.L in Fig. 4B exhibit that the currentsuppression by 1 mM trichloromethane is significant at poten-tials from −10 to +40 mV (n = 6, P < 0.05 or P < 0.01 vs. control).The concentration–response relationship of trichloromethane forinhibiting ICa.L was evaluated at +10 mV (Fig. 4C) and fitted to aHill equation. The IC50 of trichloromethane for inhibiting ICa.L was1.01 mM, and Hill coefficient was 0.9.

Voltage-dependence of steady-state activation (g/gmax) (Li et al.,1999) and availability (I/Imax) of ICa.L were evaluated in the absenceand presence of trichloromethane (Fig. 4D). The curves for g/gmax

and I/Imax were fitted to the Boltzmann function. Trichloromethaneat 2 mM had no effect on either g/gmax or I/Imax. The V0.5 of ICa.L

steady-state activation and slope factor were −3.8 ± 2.1 mV and−5.3 ± 0.7 m for control, and −4.9 ± 0.8 mV and −5.5 ± 0.2 mV for2 mM trichloromethane (n = 6, P = NS). The V0.5 of ICa.L availabilityand slope factor were −88.3 ± 1.6 mV and 5.2 ± 0.4 mV for control,

298 Y. Zhou et al. / Toxicology 2

Fig. 2. Effect of trichloromethane (chloroform) on action potentials in isolated ratventricular myocytes. A. Action potentials recorded (1 Hz) in an isolated ventricularmyocyte in the absence (control) and presence of 5 mM trichloromethane. B. Stim-ulation threshold current for triggering action potential in isolated rat ventricularmyocytes in the absence and presence of 5 mM trichloromethane (n = 6, *P < 0.05 vs.control). C. Mean values of APD90, APD50 and APD20 in the absence and presence of5 mM trichloromethane (n = 8, *P < 0.05, **P < 0.01 vs. control).

Fig. 3. Trichloromethane and IK1 in isolated ventricular rat myocytes. A. IK1

traces recorded in a representative cell in the absence and presence of 10 mMtrichloromethane. The current was evoked by voltage steps to between −120 and−20 mV from a holding potential of −40 mV. B. Current–voltage (I–V) relationshipsof IK1 in the absence and presence of 10 mM trichloromethane. The current wasmeasured from zero to the current peak (n = 6, P = NS).

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and −88.1 ± 2.1 mV and 5.6 ± 0.5 mV for 2 mM trichloromethane(n = 6, P = NS).

The recovery of ICa,L from inactivation was determined usinga paired-pulse protocol, and the recovery curves were fitted to amono-exponential equation (Fig. 4E). Trichloromethane at 2 mMsignificantly slowed the recovery of ICa.L from inactivation. The timeconstant of ICa.L recovery from inactivation was 51.0 ± 9.8 ms incontrol, and 97.7 ± 5.6 ms after trichloromethane application (n = 6,P < 0.01 vs. control).

3.5. Blockade of Ito by trichloromethane

Fig. 5A displays the voltage-dependent Ito traces recordedwith the voltage protocol shown in the inset in a representa-tive myocyte in the absence and presence of trichloromethane.Trichloromethane at 5 mM substantially reduced the current, andthe effect was partially reversed on washout. Fig. 5B illustrates theI–V relationships of Ito. Ito was inhibited by 5 mM trichloromethaneat test potentials from +10 to +60 mV (P < 0.01 vs. control, n = 6),and the inhibition was partially reversed by washout (P < 0.05 vstrichloromethane). The curve of concentration-dependent inhibi-tion of Ito by trichloromethane was fitted to a Hill equation (Fig. 5C).The IC50 of trichloromethane for inhibiting Ito was 2.4 mM with Hillco-efficient of 1.2.

Voltage-dependence of Ito steady-state activation (g/gmax)(Li et al., 2002a) and availability (I/Imax) were evaluated inthe absence and presence of trichloromethane (Fig. 5D). Thecurves for g/gmax and I/Imax of Ito were fitted to the Boltzmannfunction. The voltage-dependence of Ito activation was posi-tively shifted by trichloromethane (V0.5: 3.5 ± 0.7 mV in controlvs. 15.3 ± 1.6 mV with trichloromethane, n = 6, P < 0.01) withoutchanging slope factor (−12.7 ± 1.16 mV vs. −11.4 ± 0.7 mV withtrichloromethane). Trichloromethane had no effect on the avail-ability of Ito (V0.5: −34.4 ± 2.1 mV in control vs. −39.0 ± 1.6 mV withtrichloromethane, n = 6, P = NS).

Recovery of Ito from inactivation was determined using a paired-pulse protocol as shown in the inset (Fig. 5E) in the absenceand presence of trichloromethane, and the recovery curves werefitted to a mono-exponential equation. Trichloromethane at 5 mMdid not affect Ito recovery from inactivation. The time constantsof Ito recovery from inactivation were 23.9 ± 2.5 ms in control, and25.1 ± 1.2 ms after trichloromethane application (n = 5, P = NS).

3.6. INa inhibition

Because INa amplitude in cardiac myocytes is too large tomake a well-voltage control under physiological Na+ concen-tration, cardiac INa was usually recorded using a reduced bathNa+ concentration of 5 mM (Liu et al., 2007). We recordedINa HEK 293 cells stably expressing human cardiac Nav1.5gene under physiological Na+ concentration. Fig. 6A displaysthe time course of INa recorded in a representative cell witha 30 ms voltage step to −30 mV from a holding potential of−120 mV in the absence and presence of 10 mM trichloromethane.Trichloromethane rapidly inhibited INa, and the effect was reversedon washout. Fig. 6B shows the voltage-dependent INa tracesrecorded in a typical experiment with the voltage protocol asshown in the inset. The voltage-dependent INa was substan-tially reduced by 10 mM trichloromethane. Fig. 6C illustratesthe I–V relationships of INa before and after trichloromethaneadministration. Trichloromethane (10 mM) inhibited INa at testpotentials between −50 and 40 mV (n = 6, P < 0.05, or P < 0.01 vs.

control). The concentration-dependent inhibition of INa was eval-uated at −30 mV (Fig. 6D) and fitted to Hill equation. The IC50 oftrichloromethane for inhibiting INa was 8.2 mM, and Hill coefficientwas 2.6.

Y. Zhou et al. / Toxicology 290 (2011) 295–304 299

Fig. 4. Inhibition of ICa.L by trichloromethane (chloroform) in isolated rat ventricular myocytes. A. ICa,L traces recorded in a representative myocyte in the absence and presenceof 10 mM trichloromethane. The current was determined with 300 ms voltage steps to between −30 and +50 mV from a holding potential of −50 mV as shown in the inset.B. I–V relationships of ICa.L (n = 6, *P < 0.05, **P < 0.01 vs. control). The currents were measured from peak current to the current level at end of depolarization voltage steps. C.Concentration–response relationship of trichloromethane was fitted to a Hill equation in rat ventricular myocytes (n = 4–7 for each concentration point). D. Voltage-dependentactivation variable (g/gmax) was calculated with I–V relationships in B. Availability (I/Imax) of ICa,L was recorded with a 300 s voltage step after 1 s preconditioning voltagesbetween −90 and +10 mV, then to +10 mV (30 ms) from a holding potential of −80 mV in the absence and presence of 2 mM trichloromethane. The curves were fitted to aBoltzmann equation to obtain the V0.5 of voltage-dependent activation and availability. E. Recovery time course of ICa,L from inactivation in the absence (control) or presenceof 2 mM trichloromethane was determined with the protocol as shown in the inset. The curves were fitted to a mono-exponential equation.

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Voltage-dependence of steady-state activation (g/gmax) (Lit al., 2002b) and availability (I/Imax) of INa were evaluated inhe absence and presence of trichloromethane (Fig. 6E). Theurves for g/gmax and I/Imax of INa were fitted to the Boltz-ann function. Trichloromethane at 10 mM had no effect on

/gmax of INa (V0.5: −38.8 ± 1.1 mV in control vs. −39.9 ± 3.3 mVn trichloromethane, n = 6, P = NS). However, the V0.5 of the INavailability (I/Imax) was negatively shifted from −88.3 ± 0.7 mV forontrol to −98.1 ± 0.9 mV with trichloromethane (n = 7, P < 0.01 vs.ontrol); the slope factor was not changed (5.2 ± 0.2 mV for control,.6 ± 0.2 mV for trichloromethane).

The recovery of INa from inactivation was determined using aaired-pulse protocol as shown in the inset (Fig. 6F) in the absencend presence of trichloromethane, and the recovery curves weretted to a mono-exponential equation. Trichloromethane (10 mM)

lowed INa recovery from inactivation. The time constants of INaecovery from inactivation were 4.6 ± 0.2 ms in control, and wasncreased to 8.9 ± 1.6 ms by trichloromethane application (n = 6,< 0.01 vs. control).

3.7. Inhibition of human HCN2 current

HEK 293 cell line stably expressing human cardiac HCN2 genewas used to determine the effects of trichloromethane on thepacemaker current to avoid the technical problem of isolating ratpacemaker cells. Fig. 7A shows the time course of HCN2 currentrecorded in a representative cell with a 3 s voltage step to −120 mVfrom a holding potential of −40 mV in the absence and presenceof 3 mM trichloromethane. Trichloromethane gradually inhibitedHCN2 current, and the effect partially recovered on washout.Voltage-dependent HCN2 current was significantly suppressed by3 mM trichloromethane (Fig. 7B).

Fig. 7C illustrates the mean values of I–V relationships of HCN2current. Trichloromethane significantly inhibited HCN2 current atpotentials of −120 to −90 mV (n = 6, P < 0.01 vs. control). It also

slowed the activation of HCN2 channel and significantly increasedthe activation time constant at potentials of −120 to −90 mV (n = 6,P < 0.05) (Fig. 7D). The voltage-dependence of activation (g/gmax)of HCN2 current was determined by normalizing the tail current

300 Y. Zhou et al. / Toxicology 290 (2011) 295–304

Fig. 5. Suppression of Ito by trichloromethane in rat ventricular myocytes. A. Voltage-dependent Ito traces were recorded in a representative cell in the absence and presenceof 5 mM trichloromethane. The current was determined with a 30 ms step to −40 mV to inactivate INa current, followed by 300 ms steps to between −30 and +60 mV from aholding potential of −80 mV as shown in the inset. B. I–V relationships of Ito in the absence and presence of 5 mM trichloromethane (n = 6). The current was measured fromthe peak to the ‘quasi’ steady-state level (*P < 0.05, **P < 0.01 vs. control; #P < 0.01 vs. trichloromethane). C. Concentration–response relationship for the inhibition of Ito bytrichloromethane was fitted to a Hill equation (n = 4–8 for each concentration point). D. Voltage-dependent activation variable (g/gmax) was calculated with the data in B.A fittedo o fromp

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vailability (I/Imax) of Ito was determined with the inset protocol. The curves weref g/gmax was positively shifted by trichloromethane. E. Recovery time course of It

resence of 5 mM, and fitted to a mono-exponential equation.

efore and after application of 3 mM trichloromethane. The activa-ion curves were fitted to a Boltzmann equation (Fig. 7E). The V0.5f HCN2 activation was significantly shifted from −91.1 ± 1.5 mVor control to −96.2 ± 1.1 mV with 3 mM trichloromethane (n = 6,< 0.05 vs. control); slope factor was not affected (5.6 ± 0.4 mV forontrol and 4.7 ± 0.5 for trichloromethane).

The concentration–response relationship for the inhibition ofCN2 current by trichloromethane between 0.3 and 10 mM wasvaluated at −120 mV. The IC50 for inhibiting HCN2 current was.3 mM with a Hill coefficient of 1.7 (Fig. 7F).

.8. Suppression of hERG channel

A previous report demonstrated that trichloromethane cannhibit hERG current (Scholz et al., 2006). To further con-rm this effect, we determined the effect of trichloromethanen hERG channel stably expressed in HEK 293 cells (Fig. 8).

he voltage-dependent hERG current was highly inhibited by0 mM trichloromethane (Fig. 8A). The concentration-dependent

nhibition of IhERG was determined with 0.3, 1, 3, 10 mMrichloromethane. The IC50 of trichloromethane for inhibiting hERG

to a Boltzmann equation to obtain the V0.5 of Ito activation or availability. The V0.5

inactivation was determined with the paired pulse protocol (inset) in absence or

tail current was 4.0 mM with a Hill coefficient of 1.9. These resultsare consistent with the previous report (Scholz et al., 2006).

4. Discussion

In the present study, we have demonstrated thattrichloromethane has arrhythmogenic effect in isolated rathearts. The ionic mechanism underlying its arrhythmogenesis islikely related to the inhibition of ICa.L, Ito, INa, HCN2, and hERGchannels, but not IK1. The order of magnitude of ion channelinhibition by trichloromethane is ICa.L > Ito > HCN2 > hERG > INa.

It is generally accepted that trichloromethane-induced arrhyth-mia is related to the increased sensitivity of the heart to endogenouscatecholamine (Muller et al., 1997; Trochimowicz et al., 1976).Muller and colleagues demonstrated that in a conscious rat modelthe oral administration of trichloromethane slowed heart rate,increased the PR interval in ECG, induced premature ventricu-

lar beats, decreased ventricular contractile pressure velocity, andreduced intracellular Ca2+ transient amplitude in single ventricularmyocyte (Muller et al., 1997). The present study has demonstratedthe novel mechanism of cardiac toxicity of trichloromethane. This

Y. Zhou et al. / Toxicology 290 (2011) 295–304 301

Fig. 6. Effect of trichloromethane on INa. A. Time-course of INa recorded in a representative HEK 293 cell stably expressing human Nav1.5 gene with a 30 ms step to −35 mVfrom a holding potential at −120 mV as shown in the inset in the absence and presence of 10 mM trichloromethane. The current was measured from zero level to inwardpeak. Original current traces at respective time points were shown in the right. B. Voltage-dependent INa traces recorded in a typical experiment during control and afterapplication of 10 mM trichloromethane with 30 ms voltage steps to between −80 to +50 mV from a holding potential of −120 mV as shown in the inset. C. I–V relationshipsof INa in the absence and presence of 10 mM trichloromethane (n = 6, *P < 0.05, **P < 0.01 vs. control). D. Trichloromethane inhibited INa in a concentration-dependent mannerin Nav1.5-HEK 293 cells (n = 5–7 for each concentration point), and the curve was fitted to a Hill equation. E. Voltage-dependent activation variable (g/gmax) was calculatedw et). ThB y. F. Rp o a mo

cwppatiis

pI

ith the data in C. Availability (I/Imax) of INa was determined with the protocol (insoltzmann equation to obtain the V0.5 of voltage-dependent activation or availabilitrotocol (inset) in the absence or presence of 10 mM trichloromethane, and fitted t

ompound can induce cardiac arrhythmias in isolated rat heartsithout application of catecholamine, which indicates a directroarrhythmic potential. Trichloromethane reduced heart rate,rolonged PR interval of ECG, inhibited cardiac contractile function,nd caused ventricular fibrillation in isolated rat hearts. The nega-ive chronotropic effect and reduced A–V conduction observed insolated rat hearts suggests that trichloromethane directly inhibitsonic currents that participate in controlling pacemaker activity,

uch as the ICa.L and If (encoded by HCN2 and HCN4).

The shortened APD and the triangulated action potential mor-hology after trichloromethane application suggest a decrease in

Ca.L, which may contribute at least in part to the reduced heart

e curves in the absence and presence of 10 mM trichloromethane were fitted to aecovery time course of INa from inactivation was determined with the paired-pulseno-exponential equation to obtain recovery time constant.

rate. In addition, the increased threshold current for triggeringaction potentials by trichloromethane suggests a decrease in INa.This may contribute to the reduced conduction velocity that favorsre-entrant excitation and ventricular fibrillation as well as reducedheart rate. Reduction of ICa.L in rat ventricular myocytes may beresponsible for the triangulated action potential and at least in partfor the decreased heart rate and contractile function in isolated rathearts and reduced Ca2+ transient in cardiac myocytes (Muller et al.,

1997).

It is well recognized that the cardiac pacemaker current Ifis encoded by the hyperpolarization-activated cyclic nucleotide-gated channels (i.e. HCN2 and HCN4), which are activated by

302 Y. Zhou et al. / Toxicology 290 (2011) 295–304

Fig. 7. Inhibition of HCN2 current by trichloromethane. A. Time-course of HCN2 current recorded in a representative HCN2-HEK 293 cells with a 2 s voltage step to −120 mVfrom a holding potential at −40 mV (inset) in the absence and presence of 3 mM trichloromethane. The current was measured from the zero to the current level at end ofvoltage steps. Original current traces at corresponding time points are shown in the right panel. B. Voltage-dependent HCN2 current traces recorded in a representative cellwith 2 s voltage steps to between −120 and −40 mV, from a holding potential of −40 mV in the absence and presence of 3 mM trichloromethane. C. I–V relationships of HCN2current in the absence and presence of 3 mM trichloromethane. (n = 6, **P < 0.01 vs. control). D. Voltage-dependent activation time constant (tau) of HCN2 current in theabsence and presence of 3 mM trichloromethane (n = 6, *P < 0.05, **P < 0.01 vs. control). E. Activation conductance curves of HCN2 current measured from the zero level to thepeak of tail current in the absence and presence of 3 mM trichloromethane were fitted to a Boltzmann equation. Trichloromethane negatively shifted the activation curve. F.C ted at

cItoT

oncentration–response relationship of trichloromethane for inhibiting was evalua

yclic AMP (Barbuti and DiFrancesco, 2008; Milanesi et al., 2006).

n the present study, we demonstrated for the first time thatrichloromethane significantly inhibited cardiac HCN2 current,ne of the dominant subtypes of native If in human hearts.richloromethane negatively shifted the activation curve of HCN2

−120 mV in HCN2-HEK293 cells (n = 5–7 for each concentration point).

current, and decreased inward current amplitude at diastolic

potentials. The reduction of HCN2 current may also partiallyaccount for the decreased heart rate and prolonged PR intervalin ECG. This action is independent from the catecholamine-sensitization with trichloromethane. If this is the case, the

Y. Zhou et al. / Toxicology 290 (2011) 295–304 303

Fig. 8. Effect of trichloromethane on hERG current. A. hERG current traces recorded in a representative hERG-HEK 293 cell in the absence and presence of 10 mMtrichloromethane. The current was determined with 3 s voltage steps to between −60 to +60 mV from a holding potential of −80 mV, followed by a step at −50 to recordthe current tail as shown in the inset. B. I–V relationships of hERG current measured from zero level to the peak of tail current in the absence and presence of 1 mMt relatit

�H

mcmebreiNpTgr1va1

thTaaihpps

richloromethane (n = 6, *P < 0.05, **P < 0.01 vs. control). C. Concentration–responseo a Hill equation in hERG-HEK 293 cells (n = 6 for each concentration point).

1-adrenoceptor-mediated increase of cyclic AMP would enhanceCN2 current.

INa is responsible for initiation of action potentials in cardiacyocytes (Roden et al., 2002). Inhibition of Na+ channels is asso-

iated with various cardiac arrhythmias in clinical and in animalodels, such as long QT syndrome and Brugada syndrome (Bezzina

t al., 1999; Wang et al., 2000). It is generally accepted that inhi-ition of cardiac INa reduces conduction velocity, which promotese-entry arrhythmias (Roden et al., 2002; Schott et al., 1999; Wangt al., 2002). In the present study, we found that trichloromethanenhibited human Nav1.5 channel, a dominant subtype of cardiaca+ channels, by shifting the inactivation curve to hyperpolarizingotentials and slowing the recovery of channel from inactivation.hese effects account for the increased threshold current for trig-ering action potential. Similar inhibitory effect on Na+ channel waseported for halothane, isoflurane and sevoflurane (Weigt et al.,997). In addition, the suppression of INa would reduce intra-entricular conduction, which favors the initiation of re-entrantctivity, a substrate of ventricular fibrillation (Antzelevitch et al.,999).

The transient outward K+ current Ito inhibition byrichloromethane theoretically prolongs action potential duration;owever, this effect was not observed in current clamp mode.he balance of inward Ca2+ channel and outward K+ channelre pivotal in the determination of the cardiac repolarization ofction potential (Roden et al., 2002). Therefore, inhibition of bothnward Ca2+ channel and outward K+ channels such as Ito and

ERG channel in this experiment implies modulation of actionotential in both directions. Inhibition of ICa,L was relatively moreotent than outward K+ currents in ventricular myocytes. Thehortened phase 1 and phase 2 of the action potential indicate

onship of trichloromethane for inhibiting hERG tail current (at +30 mV) was fitted

a more prominent influence of ICa,L than Ito. The IC50 (2.4 mM)of trichloromethane for inhibiting Ito is higher than that forinhibiting ICa.L (1.01 mM). In addition, we further confirmed thattrichloromethane inhibited hERG channel in a concentrationdependent manner with an IC50 of 4.0 mM in HEK 293 cells stablyexpressing hERG gene, consistent with a previous report (Scholzet al., 2006).

Collectively, in addition to the increased sensitization of theheart to catecholamine, the present study demonstrated a directproarrhythmic effect of trichloromethane, which is likely relatedto alter the cardiac action potential shape (triangulation, relatedto ICa.L inhibition), electrical impulse generation (If channel inhibi-tion), and conduction (INa inhibition).

Conflict of interest

None is declared.

Acknowledgements

The work was supported in part by a grant from Sun ChiehYeh Heart Foundation of Hong Kong. Yuan Zhou and Hui-JunWu are supported by a postgraduate studentship from the Uni-versity of Hong Kong. The authors thank Dr. G. Robertson forproviding the hERG/pcDNA3, Dr. Carol A. Vandenberg for provid-

ing the human Kir2.1/pcDNA3, Dr. J. Makielski for providing thehuman SCNA5/pcDNA3, and Dr. A. Ludwig for providing the humanHCN2/pCDNA3. We thank Mr. Chi-Pui Mok for the excellent tech-nical support.

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