Cytotoxicity in vitro of organotin compounds to fish hepatoma cells PLHC-1 (Poeciliopsis lucida)

EUEVIER

Aquatic Toxicology 32 (1995) 143-l 60

Cytotoxicity in vitro of organotin compounds to fish hepatoma cells PLHC- 1 (Poeciliopsis Zucida) Beat J. Briischweiler”, Friedrich E. Wiirglerb, Karl FenP* “Swiss Federal Institute for Environmental Science and Technology (EA WAG), ijberlandstrasse 133, CH-8600 Diibendorf; Switzerland bInstitute of Toxicology, Swiss Federal Institute of Technology and University of Ziirich. Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland

Received 1 August 1994; accepted 17 October 1994

Abstract Cytotoxicity in vitro to fish hepatoma cells PLHC-1 has been analyzed for a series of 21 organotin compounds consisting of all degrees of alkylation and arylation. The sensitivity of the neutral red (NR) assay and the tetrazolium salt reduction (MTT) assay was similar for most of the organotin compounds. Cytotoxic effects were found at concentrations between lo-* M and lo-* M. For various trisubstituted organotin compounds, including tributyltin and triphenyltin, which are used in antifouling paints and as pesticides, respectively, cytotoxic concentrations in the range of lo-’ M to 10m6M were observed. Based on the concentration reducing NR uptake by 50% (NR&, the sequence of cytotoxicity amongst butyltins was tributyltin > bis(tributyl)tin > dibutyltin > tetrabutyltin > butyltin > tin(IV). Tributyltin induced effects on cell functions quickly, as a reduction in NR uptake by 30% was recorded after 30 min exposure to 4.1 O-’ M. The ranking order in cytotoxicity in the MTT assay of phenylated organotins was triphenyltin > diphenyltin > phenyltin > tin(IV). Cytotoxic concentrations of tributyltin and triphenyltin measured in the bromodeoxyuridine (BrdU) assay and with the crystal violet (CV) staining method do not differ significantly from those determined in the NR and MTT assay. T&and disubstituted organotin compounds exhibit a significant correlation between the noctanol/water partition coefficient (logK,,) and the NR,, (n = 10, r = 0.86, P = 0.001) and the MTT,, values (n = 11, r = 0.85, P = O.OOl), respectively. In addition, good qualitative correlations between in vitro cytotoxicity data and in vivo fish toxicity data were found (for NR assay n = 8, r = 0.86, P = 0.001; for MTT assay n = 9, r = 0.80, P = 0.001). The results indicate that the in vitro cytotoxicity assays using PLHC-1 cells are useful tools for the estimation of the acute toxicity to fish of organotins and possibly other compounds. Keywords:

Neutral

Poeciliopsis lucida; Cytotoxicity; Fish hepatoma cells; Organotin compounds: red assay; MTT assay; Bromodeoxyuridine; Crystal violet; In vitro-in vivo comparison

*Corresponding author. Tel. (+41-l) 823 53 32; Fax (+41-l) 823 50 28. 0166-445X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI

0166-445X(94)00087-5

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1. Introduction A general approach to assess the aquatic toxicity of chemical substances is based on acute fish toxicity tests. Thereby, concentrations of the test chemical in water that result in 50% death of the fishes after 24, 48 or 96 h exposure are estimated. For scientific, economic and ethical reasons, in vitro models using fish cell cultures have been developed (Rachlin and Perlmutter, 1968; Kocan et al., 1979; Bols et al., 1985). Fish cell lines have been established for more than a decade and are increasingly being used for ecotoxicological studies in the last few years (Ahne, 1985; Babich and Borenfreund, 1991; Lee et al., 1993). The PLHC-1 cell line is a hepatoma cell line derived from topminnow (Poeciliopsis lucida) exposed to 7,12_dimethylbenzanthracene (Hightower and Renfro, 1988). These cells have a xenobiotic-metabolizing capacity (Babich et al., 1991) and contain an aryl hydrocarbon (Ah) receptor (Hahn et al., 1993). Furthermore, the capacity to induce cytochrome P4501A was shown after exposure to 3,3’,4,4’-tetrachlorobiphenyl (Hahn et al., 1993). Neutral red (NR) and tetrazolium salt reduction (MTT) assays belong to the most widely used endpoints for the cytotoxicity measurement of chemicals in monolayer cell cultures. The NR assay is based on the accumulation of neutral red in lysosomes of viable cells (Borenfreund and Puerner, 1985). Treatments causing membrane damage inhibit the accumulation of this dye. The MTT assay detects the reduction of the soluble yellow MTT tetrazolium salt to a blue insoluble MTT formazan product by mitochondrial succinate-dependent dehydrogenase (Mosmann, 1983; Denizot and Lang, 1986). Cells with active mitochondria are required to catalyze the reaction. Cell proliferation can be measured using a technique in which a thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU), is incorporated into replicating DNA, and subsequently localized with a specific monoclonal antibody (Gratzner, 1982). The relative protein content attached to the well bottom can be quantified by the crystal violet (CV) staining method (Saad et al., 1993). Organotin compounds find wide application in industry and agriculture. Monoand diorganotins with butyl, methyl and octyl groups are used as catalysts, thermal and UV stabilizers in polyvinyl chloride, and as catalysts in the production of polyurethane foam. Tributyltin, triphenyltin and tricyclohexyltin compounds are used as biocides (Blunden and Chapman, 1986). In recent years, special attention has been paid to tributyltins which are used as antifouling paints for ships or fishing nets. Direct entry of tributyltins into the aquatic environment leads to water concentrations that are considered to be toxic for a variety of aquatic organisms (Hall and Pinkney, 1985; WHO, 1990; Fent and Hunn, 1991). Marine organisms such as oysters (Alzieu, 1986) and gastropods (Bryan et al., 1986; Bryan et al., 1989) are negatively affected at concentrations as low as 1 rig/l,, while toxic effects were detected in zooplankton (Bushong et al., 1990) and algae (Beaumont and Newman, 1986) at concentrations between 0.1 and 2 pug/l. Reduced survival of embryos and larvae of European minnows, Phoxinus phoxinus, was found at tributyltin concentrations of 4.3 ,L@ (1.5. IO-’ M), and histologic alterations were recorded at 0.8 ,&l (2.8.10-“M) (Fent and Meier. 1992). Organotin compounds released into the aquatic environment are transformed chemically and biologically. Since degradation of tributyltin results in

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formation of dibutyltin, monobutyltin, and finally inorganic tin (Maguire et al., 1983) knowledge of the toxicities of different organotin compounds is of significant interest. Recently, Nagase et al. (1991) determined LC,, values of 29 organotin compounds for the red killifish, Oryzius Zatipes, and studied quantitative structure-activity relationships (QSARs). These data showed that LC,, values cannot be estimated from their physicochemical and topological properties alone. The goal of this work is the analysis of the cytotoxic effects of 21 organotin compounds to PLHC-1 cells using the NR and MTT assays in order to obtain their sequence of cytotoxicity, as well as the difference between the sensitivities of the BrdU and CV cytotoxicity assays. Because each assay has a different mechanistic basis, additional information on the biochemical mechanism of the chemicals could be expected. The objective was to correlate the in vitro data with the available acute in vivo data, and with n-octanol/water partition coefficients. Hence, practically relevant conclusions about the usefulness of in vitro cytotoxicity studies with fish cell cultures for the determination of the acute toxicity of chemicals to fish can be derived.

2. Materials and methods 2.1. Abbreviations BrdU, 5-bromo-2’-deoxyuridine; BSA, bovine serum albumin; CV, crystal violet; DMSO, dimethyl sulfoxide; log&,, logarithm of n-octanol/water partition coefficient; MEM, minimum essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide; NR, neutral red; PBS, phosphate-buffered saline. 2.2.

Chemicals and solutions

Tetramethyltin, tetrabutyltin, tetraphenyltin, bis(tributyltin)oxide, dimethyltin dichloride, dibutyltin dichloride, tin(IV)chlor MTT dye, BSA and Tween 20TMwere purchased from Fluka AG (Buchs, Switzerland); tripropyltin chloride, diphenyltin dichloride, and crystal violet from Merck (Darmstadt, Germany), tripentyltin chloride and tricyclohexyltin chloride were from Aldrich (Steinheim, Germany); tetraethyltin, tetrapropyltin and triethyltin chloride were from Strem Chemicals (Newburyport, USA); methyltin trichloride and phenyltin trichloride were from Johnson Matthey (Danvers, USA); butyltin trichloride was from Ventron (Karlsruhe, Germany), and dipropyltin dichloride from Pfaltz & Bauer (Waterbury, USA). Triphenyltin chloride was a gift from J. Schwaiger (Bayerische Landesanstalt fur Wasserforschung, Germany). Eagle minimum essential medium, fetal calf serum and L-glutamine were purchased from Seromed (Berlin, Germany). Trypsin was obtained from Gibco (Basel, Switzerland), penicillin G, streptomycin and NR dye were from Sigma (Buchs, Switzerland). The cell proliferation assay was purchased from Amersham (Buckinghamshire, UK).

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2.3. &II culture Fish hepatoma cells PLHC-1 were kindly supplied by L.E. Hightower (University of Connecticut, USA) and were grown and subcultivated as described previously (Hahn et al., 1993). The cells were maintained as monolayer cultures at 30°C in a 5% CO? atmosphere in Eagle MEM with Earle’s salts, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 25 mM sodium hydrogen carbonate, 100 units/ml penicillin G and 100 pug/ml streptomycin in 75cm’ flasks. For the cytotoxicity assays, individual wells of a 96-well tissue culture microtiter plate (NunclonTM) were inoculated with 100 ~1 medium containing -8 x IO4 PLHC-I cells. Cells were allowed to attach, and were grown in the incubator until a confluency of -90% was reached after l-2 days. 2.4.

Trrutment

ti’ith organotins

All organotin compounds were dissolved in DMSO in concentrations of 2 M, 0.2 M and 0.02 M, depending on the solubility of the compound. Tetraphenyltin was dissolved in toluene in a concentration of 2 mM. These stock solutions were diluted with MEM two hundred times and further dilutions were made with MEM in a 1:5 or 1 : IO ratio. The pH was adjusted to 7.4 with 0.5 N NaOH. After removal of the old medium, 100 ~llwell of test medium containing organotin compounds or MEM (controls) was added to the cells and incubated at 30°C for 24 h, or other time periods in the kinetic experiment. 2.5. Neutrul red US.YUI* The N R assay protocol was adapted from Borenfreund and Puerner ( 1985). At the end of the 24-h incubation period, the old medium was discarded and the cultures were washed with PBS. The NR solution containing 50 pug NR/ml MEM (without phenol red) was filtered through a 0.2~,um filter (Schleicher & Schuell) to remove fine precipitates and dye crystals. 200 ~1 NR solution/well was added and incubated for 3 h at 30°C. Thereafter, 100 ~1 of the fixative 3% paraformaldehyde (3% CaCl,) was added to each well for 3 min and subsequently washed with 100 ~1 1% paraformaldehyde (1% CaCl,) for 1 min. The NR dye was extracted with 100 ~196% isopropanol (4% 1 N HCI) per well for 1O-l 5 min on a plate shaker. Optical density was measured in 96-well plates at 540 nm using a microplate reader (Dynatech MR5000 UV). Cytotoxicity of tetrabutyltin, tributyltin chloride, triphenyltin chloride, and diphenyltin dichloride was determined five times in independent experiments, using six replicate wells per toxicant concentration. Reproducibility of the experiments was high (see results). Hence, the remaining substances were tested once using six replicates per substance concentration. 2.6.

MTT ussrr~~

The MTT

assay

was performed

as previously

described

(Bruinink

and Reiser,

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1991). A 5 mg/ml stock solution of MTT was prepared in phosphate-buffered saline, and diluted in MEM (without phenol red) to 0.5 mg/ml. After removal of the incubation medium, 50 ,LL~ MTT solution per well was added for 3 h at 30°C. The MTT solution was removed and the dark blue formazan product solubilized in 100 ,ul HEPESethanol [50 mM HEPES (pH 8)ethanol (1:9, v/v)] per well for 10 min. In order to ensure solubilization, plates were vigorously shaken and absorbance was measured in 96-well plates at 560 nm using a microplate reader. As with the NR assay, cytotoxicity of tetrabutyltin, tributyltin chloride, triphenyltin chloride, and diphenyltin dichloride was determined five times in independent experiments, using six replicate wells per toxicant concentration. As with the NR assay, the reproducibility of the experiments was high (see results). Hence, the remaining substances were tested once using six replicates per substance concentration. 2.7. Bromodeoxyuridine

assay

The BrdU assay was performed according to the protocol of Amersham (cell proliferation assay RPN 210). Briefly, 100 ,LL~ labelling medium containing BrdU and 5fluoro-2’-deoxyuridine (10: 1 ratio) was added to each well and incubated for 2 h. After washing three times with PBS, cells were fixed in acetic acid/ethanol for 30 min at room temperature, and wells were blocked with 100 ~13% BSA in PBS containing 0.1% (v/v) Tween 20 TMfor 15 min at room temperature. After washing three times with PBYTween 20TM, mouse anti-BrdU antibody and nuclease in 50 ~1 PBS were added to each well and incubated for 1 h at room temperature. After washing three times with PBS/Tween 20TM, peroxidase conjugated anti-mouse IgG,, in 50 ~1 PBS was then added to the wells and incubated for 30 min at room temperature. After three washing steps with PBS/Tween 20TM, 100 ~1 peroxidase substrate solution (0.27 mM 2,2’-azino-di-[3-ethyl-benzthiazoline-6-sulfonic acid], 0.01% H,O,) was added to each well. After 10 min incubation, absorbance was measured at 410 nm using a microplate reader. 2.8. Crystal violet staining Crystal violet staining was performed according to Saad et al. (1993). 100,~l of the fixative 1% paraformaldehyde (1% CaCl,) was added to each well for 1 h at 4°C. After washing with distilled water, 100 ,~l crystal violet solution containing 0.5% crystal violet, 33% ethanol, and 1% formaldehyde in distilled water, was added to each well and incubated for 10 min at room temperature. After washing three times with water, cells were dried. Crystal violet was extracted from cells with lOO@ ethanol per well, and absorbance was measured in 96-well plates at 595 nm using a microplate reader. 2.9. Data analysis and statistics

Data for the concentration-dependent cytotoxicity relationships are presented as the arithmetic mean percentage f standard deviation (s.d.) of six replicates in relation

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to control. Organotin concentrations that reduced neutral red uptake, the formation of MTT formazan, bromodeoxyuridine incorporation, and crystal violet staining by 50% are characterized as midpoint cytotoxicity values (NR5(), MTT,,, BrdUsO, CV,,). Values were calculated by a nonlinear regression analysis (the general curve fit function of KaleidaGraphTM, Abelbeck Software, 1993) using a Michaelis-Menten model [ V = (EC,,, . V,,,)l( EC,, + C)] for the concentration-effect relationship, where I” is the NR uptake, MTT reduction, BrdU incorporation, and CV staining, respectively in percent of control; EC,, the midpoint cytotoxicity value; V,,,,, the maximal NR uptake, MTT reduction, BrdU incorporation, CV staining, respectively; and C the concentration of the test chemical. Midpoint cytotoxicity values were regarded as being different at a significance level of 95% assuming the normal distributions of V with estimated variances from the fit of the Michaelis-Menten model (i.e. ]EC,,,, - EC,,,] > k.o,,,, with k = 1.96 and or,,, = -\lai + a& Differences in sensitivities between the different exposure concentrations and the different cytotoxicity assays were determined by means of an ANOVA (Scheffe’s F-Test) with significance limits < 5%.

3. Results 3.1. Inhibition qf’neutrul red uptukr und MTT reduction In the first experiment, the influence of the exposure time on NR uptake of tributyltin was assessed after 0.5 h to 4 days. Variation in treatment period was without effect at concentrations below 4 lo-’ M (Fig. 1). At higher concentrations, NR uptake was significantly decreased by exposure time and varied between -40% and 0% depending on the treatment period. Significantly higher cytotoxicity occurred at 4 10e7 M and 2 10m6M both after 1 day and 4 days exposure than for shorter exposure times (PcO.05). As a sigmoidal concentrationresponse relationship could be recorded only after an incubation period of at least 24 h, this treatment period (1 day) was chosen in the following experiments. In the second set of experiments, cells were exposed to different organotin compounds, and the cytotoxicity was evaluated by the NR and MTT assays (Table 1). Cytotoxicity of several organotins was determined five times in independent experiments using six replicate wells per toxicant concentration. Standard errors of the means (s.e.m.) of the calculated midpoint cytotoxicity values, expressed in percent of the mean value. were for tetrabutyltin 17% (NR) and 11% (MTT); for tributyltin chloride 23% (NR) and 13% (MTT); for triphenyltin chloride 15% (NR) and 14% (MTT); and for diphenyltin dichloride 26% (NR) and 21% (MTT). Because of this high reproducibility, the remaining chemicals were tested once using six replicates per substance concentration. The cytotoxicity of a series of butyltins as determined in the NR assay differed by 5-6 orders of magnitude (Fig. 2). The sequence of midpoint cytotoxicity was tributyltin > bis(tributyl)tin > dibutyltin > tetrabutyltin > monobutyltin > tin(W). The cytotoxicity ranking of a series of phenyltins in the MTT assay was triphenyl-

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---o---0.5h +2h 2 E

-4h

100

_+_.8h -+-Id

80

5

-4d

1 o-10

10’

10"

106

10'

Concentration

10s

[M]

Fig. 1. Inhibition of neutral red uptake by tributyltin chloride in PLHC-1 cells after exposure times of 0.5, 2, 4, and 8 h, and I and 4 days. Cells were incubated at 30°C with a series of dilutions (6 replicates per dilution). Data as the mean percentage of control f standard deviation (sd.).

tin > diphenyltin > monophenyltin > tin(IV) (Fig. 3). These results indicate that the sequence of the cytotoxicity of the different organotin compounds is a function of their degree of substitution following the order: trisubstituted organotins > disubstituted organotins = tetrasubstituted organotins > monosubstituted organotins > inorganic tin. Amongst the trisubstituted organotins tricyclohexyltin is the most cytotoxic compound (NR,, = 3.9. lo-* M) followed by tributyltin, tripropyltin, triphenyltin, tripentyltin, and bis(tributyltin) (Table 1). A significantly lower cytotoxicity was recorded for triethyltin (NR,, = 3.1 . 10m6 M). The cytotoxic sequence of disubstituted organotins, determined by the inhibition of NR uptake, started with a group of diphenyltin, dibutyltin, and dipropyltin with NR,, values around 2. lo-’ M. The NR,,, of dioctyltin and dimethyltin was 5.1 . 10m4 M and 2.2. lo-” M, respectively. Cytotoxic effects of tetrasubstituted organotins tetraethyltin, tetrapropyltin and tetrabutyltin were assessed between 10e5 M and 10m3M. In the case of monosubstituted organotin compounds, only the midpoint cytotoxicity value of phenyltin could be determined. Due to low solubility, midpoint cytotoxicities could not be determined for tetraphenyltin, tetramethyltin, methyltin, and tin(IV)chlor A good correlation between the cytotoxic values of the NR assay and MTT assay was found (n = 14, r = 0.97, P = 0.0001) (Fig. 4). In the case of triethyltin, the NR assay was about 10 times, in the case of tetrapropyltin, tripropyltin, tributyltin, and tricyclohexyltin about 5 times more sensitive than the MTT assay.

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Table 1 In vitro cytotoxicity data logarithmic n-octano]/water

for PLHC-1 cells, in vivo toxicity data for red killifish partition coefficients (logK,,) for organotin compounds

Chemical I 2 4 5 6

8 9 10 11 I2 13 14 15 I6 17 18 I9 20 21

tetramethyltin tetraethyltin tetrapropyltin tetrabutyltin tetraphenyltin triethyltin chloride tripropyltin chlortde tributyltin chloride bis(tributyltin)oxide tripentyltin chloride triphenyltin chloride tricycloyhexyltin chloride dimethyltin dichloride dipropyltin dichloride dibutyltin dichloride diphenyltin dichloride dioctyltin diacetate methyltin trichloride butyltin trichloride phenyltin trichloride tin(lV)chlo

NR_

‘l.h

>o

MTT,,,” h

> IO_’ 5.8’ 5.2, 1, I >

3.60. IO-’

IO_’ lo-’ lomJ IO-’

3.1 lo-” 1.6, 10mT 1.1 lo-2.2, IO ’ 2.9, IO.‘ 1.7. IO_’ 3.9. IO_’ 2.2. IO_‘ 2.0’ IO_‘ 2.0’ lo-‘ 1.5.10~’ 5.1.10 i > IO i > IO_-‘ > IO_’

1.1 I. lo-; I.21 I()_’ I .66’ IO-’

and uystul

-o.69c 0.93’ 3.83” 2.29’

1.91. lo-’ 8.89. IO-’

3.38” 4.30 -3.10’ 0.41’ I .49g I .40d ’

1.35’ lo-” 3.61 lo-”

-3.10’ 0.35’ 1.15”’

2.73. IO-’

> I()_‘

assq

3.4se

5.85. IO-” l._5o.1o-i 9.38. lomh

~--

not determined: >. limit of solubihty in culture medium. “;loncentration in mol/l: h 24-h exposure: ‘48-h exposure; “Data from Nagase et al. (1991): ‘Data from Jow and Hansch. unpublished results, in Hansch ‘Calculated by the z method from Rekker (1977): g Data from Vighi and Calamari (1985); ” Data from Tsuda et al. ( 1990); Data from Wulf and Byington ( 1975).

3.2. Bromodeo.uvuridinP

Oriziczs latipes, and

and Leo

(1979);

siolet .stuining

In addition to the NR and MTT assays, two additional methods were evaluated for the cytotoxicity estimation. In the BrdU assay cell proliferation is measured by the incorporation of the thymidine analogue bromodeoxyuridine into DNA, whereas the crystal violet (CV) staining method determines the cellular proteins attached to the well bottom. Cells were exposed to tributyltin and triphenyltin for 1 day and 4 days to derive kinetic data, and effects were simultaneously measured by CV staining, BrdU and NR assays (Table 2). The sensitivity of all assays was similar after l-day and 4-day exposures. The BrdU assay, however. was significantly more sensitive

B. J. Briischweiler et al. I Aquatic Toxicology 32 (1995) 143-160

140,

T

T

--a--. + + ---+--++ -+-

151

tin(lV)chlo butyltin trichloride dibutyltin dichloride tributyltin chloride bis(tributyltin)oxide tetrabutvltin

80-60--

Fig. 2. Concentration-response curves of a series of butyltins in PLHC-1 cells. Cells were incubated at 30°C for 24 h with a series of dilutions (6 replicates per dilution) and cytotoxicity was determined by inhibition of NR uptake. Data as the mean percentage of control f s.d.

compared to the CV and NR assays after l-day exposure to triphenyltin. The BrdU,,, values did not decrease significantly between 1 day and 4 days, but in the case of triphenyltin, the CV,, and NRSo values were lower after 4-day exposure than l-day exposure. This indicates that an increased treatment period from 1 day to 4 days affected the cytotoxicity of triphenyltin, whereas for tributyltin this effect was not so pronounced. 3.3. Correlation with in vivo data and K,,,,. The NR,, and MTT,, values in PLHC-1 cells can be compared with published 48-h LC,, data for the red killifish Oryzias latipes (Nagase et al., 1991; Table I) and the corresponding correlations are shown in Fig. 5. For the compounds which showed highest cytotoxicity, i.e. tributyltin, bis(tributyltin), and triphenyltin, a good correlation exists between in vivo and in vitro data. This is also the case for the less toxic chemicals tetrabutyltin, tripropyltin, dibutyltin, and phenyltin. However, the in vivo toxicity of dimethyltin is underestimated by the in vitro cytotoxicity assays, whereas the effect of diphenyltin is overestimated. Finally, the same sequence of toxicity as a function of the degree of substitution was found both in red killifish (Nagase et al.. 1991) and in this study.

152

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-+--+-

160

trbhenvltin chloride tetraphenyltin

140-120-lOO--

80 -60 -40-20-O--

m’l/

1-T1

I 1r““11 , ’ / 5““‘I ’ V”‘S?

109

10’0

10-s

10-T

106

Concentration

I 7 c “‘1’1 10s

“‘,“I I r r “+ 104

1 o-3

[Ml

Fig. 3. Concentration-response curves of phenyltins in PLHC-I cells. Cells were incubated at 30°C for 24 h with a series of dilutions (6 replicates per dilution) and cytotoxicity was determined by inhibition of MTT reduction. Solubility of tetraphenyltin was limited to IO-’ M. Data as the mean percentage of control k s.d.

Published and calculated K,, values, which are given in Table 1, were correlated with the corresponding NR,, and MTT,,, values (Fig. 6). Tetra- and monosubstituted organotins were excluded from this correlation, because no midpoint cytotoxic values could be determined (methyltin, butyltin), or accurate K,, values were not available (tetraethyltin, tetrapropyltin, tetrabutyltin, tetraphenyltin). The midpoint toxicity

Table 2 Comparison of CV staining, BrdU assay and NR assay 4 days exposure to PLHC-1 cells CV,,,”

Chemical

tributyltin chloride triphenyltin chloride

for tributyltin

and triphenyltin

BrdU,,,“

after

1 day and

NR,,,”

1 day

4 days

1 day

4 days

1 day

4 days

4.6 10-j 6.3 IO-’

2.4. It.-’ 1.5~10~:

2.7. 1omY 2.0. 10YY

2.1 10~’ 1.2.10~~

3.0. to-:

2.0. lo-’ 2.0. IO_’

“Midpoint cytotoxicity values determined by crystal violet staining (BrdU,,). and neutral red assay (NR,,,): concentration in mol/l.

5.1 lo-;

(CV,,,). bromodeoxyuridine

assay

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6

Fig. 4. Correlation between NR,, and MTT,, values for 14 different organotin exposure with linear regression equation: log(l/MTT,,) = 0.94.1og(l/NR,,)-0.08 P = 0.0001). Compound numbers relate to Table 1.

7

8

compounds after 24 h (n = 14, r = 0.97,

values derived from both assays showed a significant correlation with K,, values (for NR,, values n = 10, Y= 0.85, P = 0.001; for MTT,, values n = 11, r = 0.85, P = 0.001). Hence, the K,, values of disubstituted and trisubstituted organotin compounds with up to six C atoms per substituent may serve as suitable predictors of cytotoxicity.

4. Discussion Detectable cytotoxic effects of organotin compounds on PLHC-1 cells occurred in a broad range of concentrations from lo-* M to 10m2M. Trisubstituted organotin compounds were the most cytotoxic, followed by disubstituted and tetrasubstituted organotins. The lowest cytotoxicity was detected for monosubstituted organotins and inorganic tin. A qualitative correlation of these cytotoxicity values with acute LC,, values for red killifish has been shown in Fig. 5. Also, a significant correlation between K,, values and cytotoxicity values has been recorded for tri- and disubstituted organotins (Fig. 6). 4.1. Comparison with other in vitro cytotoxicity data

In the present study, cytotoxic effects of organotin compounds to hepatoma cells

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7 -, 6 -I-

5 :-

4 1’

3 1?

3

4

5 &l(l

6

7

8

/LC,,)

Fig. 5. Correlation in vitro midpoint regression linear

between in vivo LC,, values in red killifish Oryzias lutipes (Nagase et al., 1991) and cytotoxicity values (EC,,) determined in the NR and MTT assays (this study) with equations: log(llNR,,) = 1.I 1.log(l/LC,,)-1 .OS (n = 8, r = 0.86, P = 0.001); log(IIMTT,,) = 0.89.log( l/LC,,)-0.07 (n = 9. I = 0.80, P= 0.001). Data given with arrows mean that cytotoxicity values could not be determined because of limited solubihty. Arrows point to the direction of the expected values. Compound numbers relate to Table 1.

PLHC-1 were assessed employing the NR and MTT assays. Babich and Borenfreund (1988) used the NR assay to evaluate the cytotoxicity of organotin compounds towards fibroblasts of bluegill sunfish (BF-2 cells). For tributyltin and dimethyltin, the cytotoxic effects occurred at a similar concentration as in this study (Fig. 7). Whereas the cytotoxicity of diphenyltin and dipropyltin was 2-3 orders of magnitude lower than in PLHC-1 cells, it was higher for dibutyltin and tetrabutyltin. In BALB/c mouse 3T3 fibroblasts, the cytotoxic concentration range of organotins was smaller than in PLHC-1 cells (Fig. 7). NR,, values between 4. lo-’ M and 3 . 1O-6 M were reported for trisubstituted organotins (Borenfreund et al., 1988). The ranking of the cytotoxic potencies was triphenyltin hydroxide > tripropyltin chloride > tributyltin chloride > tricyclohexyltin bromide > triethyltin bromide > trimethyltin hydroxide. The sequence of cytotoxicity in the NR assay for a series of tri- and disubstituted organotins towards murine neuroblastoma cells N,a was the same as in 3T3 fibroblasts (Borenfreund and Babich, 1987). The concentration range of the midpoint cytotoxicity values for disubstituted organotins was lower than in PLHC-1 cells (lo-’ M and 10e5 M), but the sequence of cytotoxicity was similar to that in the fish cell line. Comparison of the in vitro data of cells from different tissues (e.g. thymocytes, hepatocytes, fibroblasts, neuroblastomas) and different species (e.g. rat, mouse, hamster, fish) leads to the conclusion that the sequence of cytotoxicity of organotins is

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2

3

4

5

WLv Fig. 6. Correlation between the logarithm of the n-octanollwater partition coefficients (K,,.) and NR,,, as well as MTT,, values (EC,,) with linear regression equations: log(l/NR,,) = 0.57.logK,, + 4.90 (n = 10, + 4.38 (n = 11, r = 0.85, P = 0.001). Compound numr = 0.85, P = 0.001); log(l/MTT,,) = 0.54.logK,, bers relate to Table 1.

very similar independent of the cell origin, animal species, and the used endpoints. The fish hepatoma cell line PLHC-1, the mouse fibroblast cell line 3T3, and the murine neuroblastoma cell line N,a reacted most sensitively and similarly to organotins. 4.2. Relationships

with in vivo data and K,,,

The in vitro data from PLHC-1 cells and the in vivo data from red killifish (Nagase et al., 1991) correlate (Fig. 5). Other LC,, values of organotins towards other freshwater fish species also show correlations between the in vitro and in vivo data. Leeuwangh et al. (1976) found the same sequence of toxicity for phenyltins in guppies as in the present study, namely triphenyltin chloride > diphenyltin dichloride > phenyltin trichloride > tin(IV)chlor Plum (1981) observed 48-h LC,,, values of bis(tributyltin)oxide, dibutyltin dichloride, and butyltin trichloride at 1.7 . lo-’ M, 3.3. 10m6M, and 2.8. 10m4 M, respectively, in golden orfe (Leuciscus idus melanotus). In addition to fish, the acute toxicity of organotin compounds has been studied for water flea (Daphnia magna) (Vighi and Calamari, 1985), marine crab larvae (Rhithropanopeus harrisii) (Laughlin et al., 1985) green and blue-green algae (A&istrodesmus #catus, Scenedesmus quadricauda, Anabaena jlos-aquae) (Wong et al., 1982). In these studies, the sequence of toxicity, based on the midpoint cytotoxicity values, was trisubstituted organotins > disubstituted organotins = tetrasubstituted

156

2

3

4 log(l/NRso

5

6

7

8

PLHC-1)

Fig. 7. Correlation between NR,,, values oforganotin compounds obtained in fish hepatoma cells PLHC-1 (this study), fish fibroblasts BF-2 (Babich and Borenfreund, lY88), and mouse fibroblasts 3T3 (Borenfreund and Babich, 1987: Borenfreund et al.. 1988). log( IINR,,, BF-2) = 0.97.log(l/NR,,, PLHC-I)-0.57 (n = 6. I = 0.63, P = 0.001); log(l/NR,, XT) = 0.61 .log( I/NR,,, PLHC-I) + 2.78 (n = 9. r = 0.90, P = 0.001). Compound numbers relate to Table I.

organotins > monosubstituted organotins with partial overlap of some of the classes. These results are in agreement with the in vitro cytotoxicity data in PLHC-1 cells and the in vivo acute toxicity data for the red killifish (Nagase et al., 1991). In the present study, the midpoint cytotoxicity values of tri- and disubstituted organotin compounds show a good correlation with the corresponding K,, values (Fig. 6). Such a relationship was observed previously in fibroblasts of the bluegill sunfish (Babich and Borenfreund, 1987), in mouse fibroblasts and murine neuroblastoma cells (Borenfreund and Babich, 1987; Borenfreund et al., 1988), in algae (Wong et al., 1982), daphnia (Vighi and Calamari, 1985), mud crab (Laughlin et al., 1985) and fish (Nagase et al., 1991). Several attempts have been made to correlate the toxicity of organotins with physicochemical parameters other than the noctanol/water partition coefficient K,,,. Total surface area (TSA) (Laughlin et al., 1985), electronic (pKa), and steric (‘X) characteristics (Vighi and Calamari, 1985) and several topological indices (Nagase et al., 1991) have been applied, but no significant improvement of the corresponding correlations could be obtained. Thus, the hydrophobicity of these organotins seems to control toxicity to a significant extent. Results from this and other studies (Vighi and Calamari, 1985; Nagase et al., 1991) however, indicate that other factors are important as well; despite their higher hydrophobicity, tetrasubstituted organotins are found to be less toxic than trisubstituted organotins.

B. J. Briischweiler

er al. I Aquatic

4.3. Mode of action and comparison

Toxicology

of cytotoxicity

32 (1995)

143-160

157

assays

Several modes of action of organotin compounds have been described (for review, see Snoeij et al., 1987). Different types of effects on mitochondria could be distinguished (Aldridge, 1976). Trisubstituted organotins disturb the proton gradient, bind to a component of the ATP synthase complex that leads to inhibition of the ATP production, and cause a nonenergy-dependent swelling of rat liver mitochondria (Wulf and Byington, 1975). Dialkyltin compounds inhibit the two cl-keto acid oxidizing enzyme complexes in mitochondria, namely the pyruvate and a-ketoglutarate dehydrogenase. Membrane-damaging properties of trisubstituted organotin compounds towards various cell species were observed. Membrane integrity of rat bone marrow cells and rat thymocytes was affected at 10mhM and lo-’ M (Snoeij et al.. 1986). Tributyltin increases cytosolic free Ca” concentration by mobilizing intracellular Ca”, activating a Ca2’ entry pathway, inhibiting Ca” eIflux, and stimulating apoptosis in rat thymocytes (Aw et al., 1990; Chow et al., 1992). Recently, inhibition of cytochrome P450 monooxygenases in fish has been found in vitro and in vivo (Fent and Stegeman, 1993). Antiproliferative effects of trialkyltin compounds were also described for rat thymocytes (Snoeij et al., 1986) murine erythroleukemic cells (Zucker et al., 1992), and Baby Hamster Kidney cells (BHK-21 C13) (Reinhardt et al., 1982) at concentrations between 10e7 M and 10mhM. In PLHC-1 cells, this difference between antiproliferative effect (BrdU assay) and membrane damage (NR assay) was only significant for triphenyltin after 1 day exposure, but not after 4 days, or for tributyltin (Table 2). Each of the four methods used in this work has a different endpoint and may be indicative for different mechanisms of acute toxicity. While the NR uptake is a measure of cell membrane integrity, the MTT assay gives hints about the mitochondrial activity. The BrdU assay determines DNA synthesis, and CV staining yields the relative protein content. The present study shows that there are no significant differences between cytotoxicity data measured using different endpoints. This is consistent with a study where the midpoint cytotoxicity values of a series of trisubstituted organotins were found to be both of the same order of magnitude in the NR and MTT assay (Borenfreund et al., 1988). Although no immediate conclusions about the mode of action of organotin compounds can be drawn from these cytotoxicity assays, the inhibitions of BrdU/thymidine incorporation, NR uptake, and MTT reduction belong to the most sensitive effects of trisubstituted organotins found in vitro. They are in the same range as the inhibition of oxidative phosphorylation (Aldridge, 1976). or apoptosis of thymocytes (Aw et al., 1990). 4.4. Conclusions Toxic effects of different organotins on fish hepatoma cells PLHC-1 were detected and quantified in a very broad concentration range. A correlation between in vitro cytotoxicity data and acute toxicity data in fish was found. The findings corroborate the usefulness of in vitro cytotoxicity assays using fish cell cultures as preliminary screening tools for assessments of chemical risks to aquatic organisms. Since the NR

assay, the MTT assay and the CV staining method are less time-consuming and less expensive than the BrdU assay, but yield similar results, they appear well-suited for such purposes. Finally, the methods described here show also good promise for prescreening of chemicals prior to whole animal testing.

Acknowledgements We thank J. Hunn and M. Elmer for excellent technical collaboration, L.E. Hightower and J. Ryan (University of Connecticut) for kindly providing and shipping the PLHC-1 cells, W. Meier (University of Bern) and B. Saad (ETH and University of Ziirich) for helpful comments, T. Bosma and M. Gloor for support in data analysis. A. Bruinink (ETH and University of Ziirich) and R. Briischweiler (Scripps Research Institute) for reading the manuscript. and the anonymous reviewers for valuable suggestions. This work was supported by a grant from the Foundation for Reduction, Refinement, and Replacement of Animal Experiments to K. Fent (Stiftung 3R, Bern).

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