Introduction

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Chloroform, an industrial solvent and one of the most common environmental contaminants, produces carcinogenic effects in the liver and kidney of rodents (Golden et al., 1997; Lilly et al., 1997). For example, chloroform was found to induce toxicity and cell proliferation after a single gavage administration of chloroform to rats (Templin et al., 1996; Miyagawa et al., 1998). Furthermore, chloroform increased the frequency of micronucleated kidney cells in rats (Robbiano et al., 1998) and cytolethality in freshly isolated hepatocytes from mice and rats (Ammann et al., 1998). Clinically, chloroform was used as an anesthetic agent from 1847 through 1976 (Wawersik, 1997). In vitro, chloroform was found to activate inhibitory synaptic K⁺ channels in molluscan pacemaker neurons, leading to hyperpolarization (Lopes et al., 1998; Patel et al., 1999), and to modulate the gating kinetics of Shaker K⁺ channels (Correa, 1998). Understanding the mechanisms of chloroform toxicity may help to understand the mechanisms of its carcinogenicity.

In many biochemical assays, chloroform is a useful solvent. Chloroform is routinely used in HPLC (Barbas et al., 1997) and in the extraction of lipids or lipid-soluble membrane components (Nunez and Garcia-Sancho, 1996). In toxicological and pharmacological studies performed in cells, many drugs are dissolved in chloroform as stock solutions before being diluted into the cell bath in a final concentration of as much as 24 to 248 mM [0.2-2% (v/v)]. However, the possible effect of chloroform on cells is usually neglected.

In many cellular responses, an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) plays a key role (Clapman, 1995; Berridge, 1997). [Ca²⁺]_i may increase on stimulation as a result of Ca²⁺ entry and/or Ca²⁺ release from stores. In nonexcitable cells that lack voltage-gated Ca²⁺ channels, one of the primary stores for the [Ca²⁺]_i increase is the inositol-1,4,5-trisphosphate (IP₃)-sensitive endoplasmic reticulum Ca²⁺ store (Berridge, 1993). Binding of IP₃ to its receptors on the endoplasmic reticulum causes active releasing of Ca²⁺ from the endoplasmic reticulum store. This discharge of Ca²⁺ store often triggers Ca²⁺ entry, leading to a prolonged increase in [Ca²⁺]_i and refilling of Ca²⁺ stores. This Ca²⁺ influx is termed "capacitative Ca²⁺ entry" (Putney and Bird, 1993).

The aim of the present study was to investigate the effects on [Ca²⁺]_i in intact Madin-Darby canine kidney (MDCK) cells of chloroform and other solvents that are commonly used to make drug stock solutions in biomedical experiments that are performed in cells, such as ethanol, methanol, and DMSO. We have previously shown that in this renal epithelial cell, IP₃-dependent agonists such as ATP (Jan et al., 1998a) and bradykinin (Jan et al., 1998b) increase [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum Ca²⁺ store, followed by capacitative Ca²⁺ entry. In addition, thapsigargin (Jan et al., 1999a) and 2,5-di-tert-butylhydroquinone (Jan et al., 1999b) increase [Ca²⁺]_i by directly inhibiting the endoplasmic reticulum Ca²⁺ pump without elevating IP₃ levels, leading to Ca²⁺ release followed by capacitative Ca²⁺ entry. Thus, MDCK cells were used as a model to examine the effects of chloroform on Ca²⁺ handling in nonexcitable cells.

We have found with the use of Fura-2 as a Ca²⁺-sensitive fluorescent dye that chloroform, but not the other three solvents, increased [Ca²⁺]_i in a concentration-dependent manner in MDCK cells. We established the concentration-response relationships in both the presence and absence of external Ca²⁺ and determined the underlying mechanisms of chloroform-induced [Ca²⁺]_i increase. The effects of chloroform on the [Ca²⁺]_i increases induced by ATP and thapsigargin were also explored, and the effects of chloroform on human neutrophils and T24 bladder carcinoma were measured.

	Materials and Methods

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Cell Culture. MDCK cells and T24 bladder carcinoma cells obtained from American Type Culture Collection (CRL-6253; Rockville, MD) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO₂-containing humidified air.

Isolation of Neutrophils. After the informed consent was obtained, whole blood was taken by venipuncture from a healthy human volunteer with no history of infections 2 weeks before the experiments. Blood was mixed with heparin (20 U/ml), and the erythrocytes were allowed to sediment for 50 min at room temperature after a 1:6 (v/v) Hespan/blood blend. The leukocyte-rich plasma was harvested and centrifuged at 300g for 20 min. The supernatant was aspirated and centrifuged at 2170g for 15 min to produce platelet-poor plasma. The pellet from centrifugation of leukocyte-rich plasma was resuspended in 2.5 ml of platelet-poor plasma and transferred to a 15-ml tube, where it was underlayered with 2 ml of freshly prepared 42% Percoll in platelet-poor plasma. This mixture was in turn underlayered with 2 ml of freshly prepared 52% Percoll in platelet-poor plasma. The gradients were centrifuged for 10 min at 280g. The neutrophils were collected at the 42 to 51% Percoll interface. The final cell population was determined to contain >95% neutrophils by Wright's staining. The neutrophils were 98% viable as assayed by trypan blue exclusion.

Solutions. Ca²⁺ medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 5 mM glucose. Ca²⁺-free medium contained no Ca²⁺ plus 1 mM EGTA (calculated [Ca²⁺], <0.1 nM).

Optical Measurements of [Ca²⁺]_i. Trypsinized MDCK (or T24) cells and freshly isolated neutrophils (10⁶/ml) were allowed to recover in Dulbecco's modified Eagle's medium for 1 h before being loaded with 2 µM Fura-2 AM [1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid pentaacetoxymethyl ester] for 30 min at 25°C in the same medium. The cells were washed and resuspended in Ca²⁺ medium. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer by continuously recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1-s intervals. Maximum and minimum fluorescence values were obtained by adding Triton X-100 (0.1%) and EGTA (20 mM) sequentially at the end of an experiment. [Ca²⁺]_i was calculated as described previously (Grynkiewicz et al., 1985). Our previous studies showed that trypsinized MDCK cells prepared by this protocol respond to stimulation with ATP (Jan et al., 1998a), bradykinin (Jan et al., 1998b), or thapsigargin (Jan et al., 1999a) in a similar manner as cells attached to coverslips.

Chemical Reagents. The reagents for cell culture were obtained from Life Technologies (Grand Island, NY). Fura-2 AM was from Molecular Probes (Eugene, OR). U73122 [1-(6-((17b-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione], U73343 [1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione], and aristolochic acid were from BIOMOL Research Laboratories (Plymouth Meeting, PA). The other reagents were from Sigma Chemical Co. (St. Louis, MO).

Statistical Analysis. All values were reported as mean ± S.E. of five or six experiments. Statistical comparisons were determined by using Student's paired t test, and significance was accepted at P < .05.

	Results

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Effect of Chloroform on [Ca²⁺]_i in MDCK Cells. Figure 1A shows that in Ca²⁺ medium, the resting [Ca²⁺]_i had a value of 98 ± 4 nM (n = 5), which remained stable for 350 s (trace d). At concentrations between 24 and 248 mM, chloroform concentration dependently increased [Ca²⁺]_i (traces a-c). At 12 mM, chloroform had no effect. The effects of a higher concentration of chloroform were not examined. Over a time period of 5 min, the [Ca²⁺]_i signal contained a slow initial rise and an elevated phase. For example, at a concentration of 93 mM, chloroform induced a [Ca²⁺]_i increase that reached a net maximum 270 ± 6 s (n = 6; p < .05) later at a net value of 278 ± 10 nM (trace b; n = 6; P < .05), followed by a sustained phase. The rise of the Ca²⁺ signal was slower in response to lower concentrations of chloroform.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effect of chloroform on [Ca2+]i in Fura-2-loaded MDCK cells. A, concentration-dependent effects of chloroform. The concentration of chloroform was 248 mM in trace a, 93 mM in trace b, 24 mM in trace c, and 12 mM in trace d. The experiments were performed in Ca2+ medium. B, similar to A except that the experiments were performed in Ca2+-free medium. C, chloroform (93 mM)-induced changes in the 340- and 380-nm excitation wavelength signals. D, concentration-response plots of chloroform-induced responses in the presence () or absence () of external Ca2+. The y axis is the net area under the curve (30-350 s) of the [Ca2+]i increase. Data are mean ± S.E. of five or six experiments. *P < .05,  versus . Traces are typical of five or six experiments.

Internal Sources of Chloroform-Induced [Ca²⁺]_i Increase. Figure 2A shows that in Ca²⁺-free medium, the addition of 2 µM carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, induced a small [Ca²⁺]_i increase with a net peak value of 39 ± 4 nM (n = 5; P < .05), which is consistent with previous reports (Jan et al., 1998b,c, 1999a,b,c). This [Ca²⁺]_i increase most likely reflected Ca²⁺ release from mitochondria. Subsequently added thapsigargin (1 µM), an endoplasmic reticulum Ca²⁺ pump inhibitor (Thastrup et al., 1990), caused a [Ca²⁺]_i increase with a net peak value of 91 ± 5 nM (n = 5; P < .05). Chloroform (93 mM) added at 850 s still induced a [Ca²⁺]_i increase with a net peak value of 51 ± 5 nM (n = 5; P < .05). Conversely, Fig. 2B shows that in Ca²⁺-free medium, 93 mM chloroform induced a [Ca²⁺]_i increase with a net peak value of 91 ± 6 nM (n = 5; P < .05). This chloroform pretreatment abolished the [Ca²⁺]_i increase induced by subsequently added thapsigargin (1 µM) and partially decreased the [Ca²⁺]_i increase induced by 2 µM CCCP by 49 ± 5% in net peak value (n = 6; P < .05).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Chloroform-induced Ca2+ release. A, in Ca2+-free medium, CCCP (2 µM), thapsigargin (1 µM), and chloroform (93 mM) were added at 30, 400, and 900 s, respectively. B, in Ca2+-free medium, chloroform (93 mM), thapsigargin (1 µM), and CCCP (2 µM) were added at 30, 800, and 1250 s, respectively. Traces are typical of five or six experiments.

Mechanism of Chloroform-Induced Ca²⁺ Entry. In MDCK cells, depletion of Ca²⁺ stores often triggers capacitative Ca²⁺ entry (Jan et al., 1998a,b,c, 1999a,b,c). Because chloroform released Ca²⁺, we investigated whether chloroform induces Ca²⁺ influx via capacitative Ca²⁺ entry. Capacitative Ca²⁺ entry was measured by adding 3 mM Ca²⁺ to cells pretreated with chloroform in Ca²⁺-free medium. Figure 3A shows that after depleting Ca²⁺ stores for 800 s with 93 mM chloroform, the addition of Ca²⁺ induced a [Ca²⁺]_i increase with a net maximum value of 61 ± 4 nM (trace a) that was greater than control (24 ± 4 nM; trace b) by 2.5-fold (n = 6; P < .05). Thus, our data suggest chloroform-induced capacitative Ca²⁺ entry. Because La³⁺ is a potent blocker of capacitative Ca²⁺ entry in MDCK cells (Jan et al., 1998a,c, 1999a,b), we tested how La³⁺ affects chloroform-induced [Ca²⁺]_i increase. Figure 3B shows that pretreatment with 1 mM La³⁺ decreased 93 mM chloroform-induced [Ca²⁺]_i increase in Ca²⁺ medium by 44 ± 5% in the net area under the curve (30-400 s).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   A, effects of chloroform on capacitative Ca2+ entry. Capacitative Ca2+ entry was induced by depleting Ca2+ stores in Ca2+-free medium followed by the addition of 3 mM CaCl2. Trace a, chloroform (93 mM) was added at 30 s followed by CaCl2 at 800 s. Trace b, CaCl2 was added at 800 s. B, effect of La3+ on chloroform-induced [Ca2+]i rise. Trace a, control effect of 93 mM chloroform-induced [Ca2+]i rise. Trace b, similar to trace a except that 1 mM La3+ was added 10 s before chloroform. Traces are typical of five or six experiments.

Effects of Inhibition of Phospholipase C or A₂ on Chloroform-Induced Internal Ca²⁺ Release. It was shown in MDCK cells that ATP (10 µM) releases Ca²⁺ via IP₃ (Jan et al., 1998c). Figure 4A shows a representative [Ca²⁺]_i increase induced by 10 µM ATP (trace a). Incubation with U73122 (2 µM), a phospholipase C inhibitor (Thompson et al., 1991), for 220 s abolished the ATP-induced [Ca²⁺]_i increase (trace b; n = 6; P < .05). This most likely implies that U73122 had effectively blocked phospholipase C-dependent IP₃ production. Subsequently added chloroform (93 mM) induced a [Ca²⁺]_i increase with a net maximum height of 145 ± 5 nM (n = 6) that is indistinguishable from control (trace c) in the area under the curve (280-800 s) (P < .05). U73343, an inactive analog of U73122, altered neither the resting [Ca²⁺]_i nor the [Ca²⁺]_i increases induced by ATP and chloroform (not shown). Because it was shown that activity of phospholipase A₂ is linked to Ca²⁺ signaling in MDCK cells (Jan et al., 1999c), the following experiments were performed to examine the effect of inhibition of phospholipase A₂ on chloroform-induced Ca²⁺ release. Pretreatment with aristolochic acid (40 µM), a phospholipase A₂ inhibitor (Rosenthal et al., 1989), for 250 s inhibited 93 mM chloroform-induced [Ca²⁺]_i increase by 90 ± 4% in the net maximum height (trace d versus trace c; n = 6; P < .05) without altering the resting [Ca²⁺]_i.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   A, effects of inhibition of phospholipase C or A2 on chloroform-induced internal Ca2+ release. A, trace a, ATP (10 µM) was added at 30 s. Trace c, control effect of chloroform (93 mM; added at 280 s). Trace b, U73122 (2 µM) was added at 30 s followed by ATP (10 µM) at 210 s and chloroform (93 mM) at 280 s, respectively. Trace d, aristolochic acid (40 µM) was added at 30 s followed by chloroform (93 mM) at 280 s. The experiments were performed in Ca2+-free medium. Traces are typical of five or six experiments. B, effects of other solvents on [Ca2+]i in MDCK cells. Trace a, 93 mM chloroform was added at 60 s. Trace b, 2% (v/v) ethanol, methanol, or DMSO was added at 60 s. The experiments were performed in Ca2+ medium. Traces are typical of five or six experiments. C, effects of chloroform on [Ca2+]i in other cells. Chloroform (93 mM) was added at 30 s. Trace a, human neutrophils. Trace b, T24 bladder carcinoma cells. The experiments were performed in Ca2+ medium. Traces are typical of five experiments. D, experiments were performed in MDCK cells bathed in Ca2+ medium. Dotted trace, thapsigargin (1 µM) was added at 30 s. Solid trace, similar to dotted trace except that the bath contained 12 mM chloroform. Traces are typical of six experiments.

Effects of Other Commonly Used Solvents on [Ca²⁺]_i. Figure 4B shows that chloroform (93 mM) induced a [Ca²⁺]_i increase with a maximum value over 300 nM (trace a). However, at a concentration of 2% (v/v), ethanol (343 mM), methanol (459 mM), or DMSO (279 mM) did not increase [Ca²⁺]_i (trace b; n = 5; P > .05).

Effects of Chloroform on [Ca²⁺]_i in Other Cells. Figure 4C shows that in Ca²⁺ medium, chloroform (93 mM) increased [Ca²⁺]_i in T24 bladder carcinoma and human neutrophils with a maximum value of 312 ± 10 and 151 ± 9 nM, respectively (n = 6; P < .05).

Effects of 12 mM Chloroform on [Ca²⁺]_i Rises Induced by Thapsigargin and ATP in MDCK Cells. Figure 4D shows that in Ca²⁺ medium, the [Ca²⁺]_i increase induced by 1 µM thapsigargin was not altered by 12 mM chloroform (n = 5; P > .05). Similarly, the [Ca²⁺]_i increase induced by 10 µM ATP was not affected by 12 mM chloroform (not shown).

	Discussion

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This report is the first to show that chloroform increased [Ca²⁺]_i in MDCK cells, human neutrophils, and bladder carcinoma. In MDCK cells, chloroform triggered both Ca²⁺ influx and Ca²⁺ release at higher concentrations (95-248 mM) because the [Ca²⁺]_i signals measured in Ca²⁺ medium were partially decreased by Ca²⁺ removal. The rising and sustained phases were both reduced by Ca²⁺ removal, suggesting the [Ca²⁺]_i increase involved Ca²⁺ influx and Ca²⁺ release throughout the whole course of measurement. At lower concentrations (24-60 mM), chloroform mainly increased [Ca²⁺]_i by releasing Ca²⁺ because Ca²⁺ removal had no effects on [Ca²⁺]_i.

The Ca²⁺ stores for the chloroform-induced [Ca²⁺]_i signal consisted of thapsigargin-sensitive endoplasmic reticulum stores, CCCP-sensitive mitochondrial stores, and other unidentified stores. This is because that in Ca²⁺-free medium, pretreatment with 93 mM chloroform prevented 1 µM thapsigargin and 2 µM CCCP from releasing more Ca²⁺, and after pretreatment with CCCP and thapsigargin, chloroform still released a significant amount of Ca²⁺. This is interesting because most of the Ca²⁺-mobilizing agents we have tested in MDCK cells, such as ATP, bradykinin, U73122, cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone, release Ca²⁺ solely from thapsigargin-sensitive stores (Jan et al., 1998a,b,c, 1999a,b). However, we recently found that the ether lipid ET-18-OCH₃, an antitumor drug, released Ca²⁺ in a manner similar to chloroform (Jan et al., 1999c). We did not further investigate the chloroform-, CCCP-, and thapsigargin-insensitive internal Ca²⁺ stores because of the lack of selective pharmacological tools for the other stores. MDCK cells appear not to possess ryanodine-sensitive Ca²⁺ stores as we previously demonstrated that neither ryanodine nor caffeine increased the resting [Ca²⁺]_i (Jan et al., 1998b).

The question arises as to how Ca²⁺ was released on chloroform stimulation. We examined whether IP₃ mediates the action of chloroform by using U73122, a phospholipase C inhibitor, to suppress IP₃ formation. U73122 appeared to abolish IP₃ formation as ATP (10 µM) added subsequently did not increase [Ca²⁺]_i. Under these circumstances, chloroform still induced a normal [Ca²⁺]_i increase. Thus, it seems unlikely that IP₃ has a role in mediating chloroform-induced Ca²⁺ release. Conversely, phospholipase A₂ may play a significant role because inhibition of phospholipase A₂ with 40 µM aristolochic acid reduced the chloroform response by 90% without depleting Ca²⁺ stores.

We found that 93 mM chloroform activated Ca²⁺ influx mainly via capacitative Ca²⁺ entry. This is because 3 mM Ca²⁺ triggered a significant [Ca²⁺]_i increase after cells had been pretreated with 93 mM chloroform in Ca²⁺-free medium for 13 min. This is supported by the result that 1 mM La³⁺, a general Ca²⁺ entry blocker, partially inhibited 93 mM chloroform-induced [Ca²⁺]_i increase.

Collectively, in the present study, we characterized the [Ca²⁺]_i increase induced by chloroform in MDCK cells and examined the underlying mechanisms. Given the results that chloroform induced significant [Ca²⁺]_i increases in cultured and freshly isolated cells at concentrations that might be achieved in the final experimental solution used in cellular signaling studies, we suggest that the effect of chloroform should be investigated before using it as a vehicle in these studies. This caution is especially important in situations in which [Ca²⁺]_i increases or Ca²⁺ store depletion may affect the results. Furthermore, drugs are preferably dissolved in ethanol, methanol, or DMSO if possible because these solvents did not alter the resting [Ca²⁺]_i at a concentration up to 2% (v/v) in the three types of cells we tested. If chloroform is the only vehicle that can be used, we recommend that its final concentration be kept below 12 mM because our data showed that at this concentration, chloroform did not affect the resting [Ca²⁺]_i or the [Ca²⁺]_i signals induced by ATP and thapsigargin.