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Cellular effects of an anionic surfactant detected in V79 fibroblasts by different cytotoxicity tests
0887-2333/90 $3.00 + 0.00 Copyright % I990 Pergamon Press plc
Toxic. in Vim Vol. 4, No. I, pp. 9-16, 1990 Printed in Great Britain. All rights reserved
CELLULAR EFFECTS OF AN ANIONIC SURFACTANT DETECTED IN V79 FIBROBLASTS BY DIFFERENT CYTOTOXICITY TESTS V. BIANCHI* Department
and E.
FORTWNATI
of Biology, University of Padua, Via Loredan 10, I-35131 Padova, Italy (Received 10 March 1989; revisions received 12 June 1989)
Abstract-Several cytotoxicity tests were employed to detect the cellular effects of low concentrations of the anionic surfactant linear alkylbenzene sulphonate (LAS). When added to growth medium containing 5% foetal calf serum, LAS did not affect V79 cell growth, nor did it alter the permeability of cell membranes. The inactivity depended on the serum component of the medium. When treatments were carried out in serum-free saline, LAS inhibited cell proliferation, made the plasma membrane permeable to otherwise-undiffusible compounds, and reduced the uptake of tritiated thymidine. The alterations in membrane permeability were evaluated from the release of cytoplasmic molecules of different size (lactate dehydrogenase, adenine nucleotides, RNA) into the medium. The sensitivity of the spectrophotometric lactate dehydrogenase assay was inadequate for the conditions of treatment required to detect the cytotoxicity of LAS. In cultures pre-incubated with tritiated adenine instead, the release of labelled ATP pool components was time and dose dependent and allowed discrimination between levels of membrane damage causing the same degree of trypan blue staining. Also, macromolecular nucleic acids were detected outside the treated cells at doses of 4-6 mg LAS/litre, which indicated severe membrane damage.
INTRODUCTION
At the same time, both the sediments and the organisms can be altered by the detergent. Bressan et al. (1989) examined the acute, subacute and chronic effects of LAS (adsorbed onto the sediment or dissolved in water) on several marine and fluvial benthic species with a wide distribution. Our endpoints for the cellular effects of LAS were general cytotoxicity and specific parameters of membrane damage, and we were interested in detecting effects of LAS doses close to those active
Tissue culture systems have frequently been used as alternatives to in vivo tests in assessing the toxicology of detergents. As components of soaps and shampoos, detergents are generally assayed for their ocular irritation potential by the Draize test on rabbits, but reproducible and predictive in vitro cell systems have been developed for the screening of irritants (Aeschbacher et al., 1986; Borenfreund and Borrero, 1984; De Leo et al., 1987; North-Root et al., 1982; Scaife, 1985; Shopsis and Eng, 1985; Shopsis et al., 1985). Most of the work in vitro has been aimed at obtaining potency ranks comparable with those from the Draize test. We have studied the cytotoxicity of linear alkylbenzene sulphonate (LAS), an anionic surfactant present in domestic detergents, to provide a cellular basis for the in vivo effects detected in a parallel study on benthic invertebrates (Bressan et al., 1989). These authors have investigated the environmental effects of LAS once it reaches surface waters, where it is distributed between water and sediments. The nature of the sediments and the activity of the benthic organisms living on them determine the environmental fate of LAS-adsorption and/or biodegradation.
in vivo.
Several approaches are available for detecting plasma membrane damage, mostly based on variations in membrane permeability. These include the loss of dye exclusion ability (Aeschbacher et al., 1986; Scaife, 1985; Thelestam and Miillby, 1976) leakage of endogenous cell components (Lock et al., 1987a,b; Malik et al., 1983; Scaife, 1985) or the release of radioactive material from cells pre-labelled with physiological precursors or their non-metabolizable analogues (Bianchi et al., 1982; De Leo et al., 1987; Malik et al., 1983; Thelestam and Mollby, 1975a,b; Walum and Peterson, 1982). We tested trypan blue exclusion and lactate dehydrogenase (LDH) leakage, but found them to be rather insensitive indicators of damage. In particular, the LDH leakage test that employs the standard spectrophotometric method to detect LDH activity in the incubation medium appeared to be unsuitable for testing LAS toxicity in our system. A modified protocol, formerly proposed by Thelestam and Miillby (1975a) for measuring nucleotide and RNA release into the medium, allowed us to evaluate the extent of membrane damage from the nature of the released cell components.
*To whom all correspondence should be addressed. Abbreviations: [2-‘H]Ade = tritiated adenine; DMEM = Dulbecco’s modified Eagle’s medium; FCS = foetal calf serum; HBSS = Hanks’ balanced salt solution; LAS = linear alkylbenzene sulphonate; LDH = lactate dehydrogenase; OD,,,, = optical density at 260 nm; (5-3H]TdR = tritiated acid; TCA = trichloroacetic thymidine. 9
10
V. BIANCHI and E. FORTUNATI MATERIALS
AND METHODS
Chemicals. LAS (length of alkyl chain lo-12 carbon atoms, 27.14% in water) was purchased from Italian chemical industries. NADH, pyruvic acid and Triton Xl00 were from Sigma Chemical Co. (St Louis, MO, USA); tritiated thymidine ([5-3H]TdR, sp. act. 25 Ci/mmol) and adenine ([2-3H]Ade, sp. act. 22 Ci/mmol) were from Amersham International (Amersham, England). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from GIBCO BRL (Eggenstein, FRG) and foetal calf serum (FCS) from Seromed, Biochrom KG (Berlin, FRG). All other chemicals and reagents were analytical grade. Cells and culture conditions. The origin and growth characteristics of the V79 line used were those previously described (Bianchi et al., 1987). The cells were routinely grown as monolayers in IO-cm petri dishes at 37’C in a 5% CO, in air atmosphere in DMEM containing 5% heat-inactivated FCS. Cytotoxicity tests. Cells were seeded 24 or 48 hr in advance at a density of lo4 cells/well in 24-well multidishes or at a density of 2 x lo4 tells/3.5-cm dish. For LDH determinations, where more cells were needed, 1.5 x 10’ tells/6-cm dish were plated 48 hr before the experiments. After some preliminary trials (see Results), all treatments with LAS were performed in Tris-buffered Hanks’ balanced salt solution (HBSS), after rinsing the cell monolayers with prewarmed HBSS to remove all traces of FCS. All experiments were run in replicate, and repeated at least twice. On account of the influence of cell density on LAS cytotoxicity (see Results), care was taken to seed the cells always at the same time of the day, and start the treatments accordingly. Experiments with radioactive precursors were run in duplicate, and each point in the graphs is the mean of quadruplicate determinations, with a variability < 10%. Inhibition of cell growth was determined 48 hr after treatment on triplicate cultures: standard deviations were no higher than expected. Cell numbers in control and treated cultures were estimated from their total macromolecular content as follows. The medium was removed from the dishes and the monolayers washed twice with cold HBSS. The soluble nucleotide pool was extracted with 60% methanol for 30 min at 4°C and discarded. The dishes were washed once with 1 ml cold methanol and thoroughly drained before dissolving the cell layer in 0.3 M-NaOH overnight at room temperature. The optical density at 260 nm (GD,bO nm) of the NaOH fraction, which corresponds to the macromolecules left on the plates after pool extraction, is used as an index of cell number (Bianchi and Celotti, 1985). Calibration curves obtained by dissolving in NaOH V79 cultures containing different numbers of cells are linear and indicate that an 0DZ6,,“,,, of 1 corresponds to lo6 cells. To determine the inhibition of [5-‘H]TdR uptake and the level of DNA synthesis in cells treated with LAS, at the end of the treatment the cultures were re-fed with complete medium and incubated for 1 hr with 0.3 pM-[5-3H]TdR either immediately, or 1 or 3 hr later. [5-3H]TdR uptake was measured by counting the radioactivity in the soluble nucleotide pool extracted with 60% methanol as above. Macro-
molecular DNA was obtained by trichloroacetic acid (TCA) precipitation from the 0.3 M-NaOH in which the cell layer was dissolved (Bianchi et al., 1987). Aliquots were collected on glass-fibre filters and counted. The exposure to increasing doses of LAS produced increasing inhibition of [5-3H]TdR uptake into the nucleotide pool. Therefore, original values of DNA radioactivity were normalized on the basis of the percentage variations of radioactivity in the pool (Levis et al., 1978). This allowed a more accurate evaluation of the level of DNA synthesis in the treated cells. Determination of membrane damage. In all experiments trypan blue staining was used as a simple immediate method to monitor the degree of membrane permeability at the end of the treatments. In some cases the cells were stained at different times after treatment to monitor changes of membrane permeability with time. A 0.5% solution of trypan blue was added to an equal volume of the medium and the percentage of stained cells was determined within 5 min by microscopic examination. LDH release was determined by the spectrophotometric procedure of Bergmeyer and Bernt (1974) according to Malik et al. (1983). After treatment the HBSS was centrifuged at 2000 rpm for 5 min and the cell-free supernatant was removed. A 0.5% Triton Xl00 solution in HBSS was used to dissolve the cell layers and cell pellets were obtained by further HBSS centrifugation. The supernatant and the cell lysate were assayed for enzyme activity. Preliminary checks had shown that LAS does not interfere with the activity of standard LDH. Release of ATP pool components. The cultures were incubated for 1 hr with 0.5 PM [2-3H]Ade in complete medium, then repeatedly washed with warm HBSS to remove extracellular radioactivity. The cells were then treated with LAS in HBSS for l-3 hr, the incubation medium was collected, and the plates were processed for pool extraction and cell dissolving as described above. Radioactivity was measured in the medium (total and acid-precipitable), in the nucleotide pool, in total nucleic acids (NaOH fraction) and in DNA (obtained by TCA precipitation of aliquots of the NaOH fraction). Release of macromolecular RNA. To detect loss of RNA from cells exposed to LAS, the cultures were left to settle for 12 hr after seeding and then received 0.05 pM [2-3H]Ade for 24 hr. At the end of incubation the radioactive medium was removed, and plates were rinsed and chased for 24 hr in fresh non-radioactive medium before treating with LAS. After treatment the radioactivity in the treatment solution and in the different cellular fractions was determined as above. With both protocols for [2-3H]Ade incorporation, all the cultures were expected to contain the same amount of tritium, since the incubation with [2-‘H]Ade took place before the treatment with LAS. In order to have a direct check of the effectiveness of the recovery in the different plates, the sum of the radioactivity measured in the medium, the pool and the total nucleic acid fraction of each culture was routinely calculated. A 20% variation in the total radioactivity recovered/culture was the upper limit accepted.
In vitro tests for surfactant toxicity
11
Table 2. Plasma membrane damage in V79 cells treated with LAS in HBSS without FCS
RESULTS
Inhibition of cell growth
Inhibition of cell proliferation in mass culture and reduction of colony-forming ability were considered to be basic indicators of cytotoxicity. A first series of trials, carried out with LAS added to the complete growth medium in a concentration range of 1 pg to 10 mg/litre, showed no effect on cell growth. Plating efficiency in clonal cultures was reduced only above 20mg/litre, with a 50% inhibition at 30 mg/litre and complete cell loss at 50 mg/litre (not shown). The low activity of LAS in complete medium could be attributed to the serum component of the medium, which probably sequestered LAS by its binding to lipoproteins (Benoit et al., 1987). In subsequent experiments the cells were treated in HBSS, where only acute treatments (shorter than a generation time) were possible. The influence of serum was tested by comparing the inhibition of cell growth by LAS in the presence or absence of 5% FCS. Treatments were carried out 24 hr after seeding and cell proliferation was evaluated 48 hr later from the OD,,,, of cell monolayers dissolved in alkali. Table 1 shows the results of two experiments in which V79 cells were treated with 0.5-10 mg LAS/litre in HBSS with or without FCS for 4 hr. Inhibition of growth appeared from 5 mg/litre onwards and was completely abolished by the presence of FCS during treatment. Permeability of plasma membrane
To obtain an indication of cell membrane damage, V79 cells treated with LAS in HBSS without FCS were exposed to trypan blue dye. A semi-quantitative evaluation of membrane damage in cultures treated with 3-7 mg LAS/litre for 1, 2 or 4 hr is given in Table 2. Membrane permeability was monitored immediately after treatment and up to 24 hr later. Effective treatments induced irreversible permeability of cell membranes, with the exception of 7 mg/litre (1 hr) and 5 mg/litre (2 hr), which allowed a partial recovery of membrane integrity. Table I. Influence of FCS on the inhibition of cell growth by LAS dissolved in HBSS with or without FCS* A
Treatment
HBSS - FCS + LAS 0.5 mg/litre I mg/litre 2.5 mg/litre 5 mg/litre 7.5 mg/litre IO mg/litre HBSS + FCS + LAS 0.5 mg/litre I mg/litre 2.5 me/litre _/ 5 mellitre
%,~~
7.5 mg/litre IO mg/litre
Exe.
ODm nm I .70 I .40 NT NT I.36 0.08 NT 0.04 NT NT NT I .62 I .75 NT I.56
1 -A
Em. 2 %t
100
91 6 3 95 103 92
ODmnm I .97 1.61 I .48 I .48 1.41 0.95 0.02 0.02 1.87 1.82 I .88 I .86 1.82 I.81 I .83
%t -
Trypan blue staining after the end of the treatment (hr) LAS treatment (mailitre) I hr
(3) (5) (7)
2hr
(3) (5) (7)
4hr
(3) (5) (7)
0 _ * _ ++ +++ _ +++ +++
100
97 100 99 97 97 98
NT = not tested *V79 cells were incubated for 4 hr in the indicated media 24 hr after seeding. Cell growth was evaluated 48 hr later in triplicate cultures from the OD,,,, of the alkali-dissolved monolayers. iValues are given as % of controls with or without FCS.
4 _
24 _ _
++ +++ +++ +++
_ I!c +++ _ +++ +++
V79 cells were treated with LAS for the indicated times 24 hr after seeding, then exposed to trypan blue at different times after the end of the treatment. Number of stained cells (scored on triplicate plates): - = 0%, f = about IO%, + + = more than 50%. + + + = 100%.
In another experiment (Table 3), a number of doses between 3 and 7.5 mg/litre were tested with 4-hr treatments and the uptake of trypan blue was checked at three time points thereafter. Cell numbers were determined 48 hr after treatment. Again, normal cell permeability was partially restored after 4-5 mg LAS/line, while concentrations higher than 5.5 mg/litre produced irreversible damage and cell detachment, as indicated by the drop in cell density two days after treatment. Tables 2 and 3 show that permeability to trypan blue does not increase progressively: within a very narrow range of doses individual cells change from being impermeable to being fully permeable. Thymidine uptake and DNA synthesis in LAS-treated ceils
The uptake of extracellular TdR and its utilization for DNA synthesis is a complex energy-requiring process that involves many different enzymes. Inhibition of TdR uptake can thus be taken as a broad endpoint of cytotoxicity, being the possible outcome of many individual mechanisms. We examined the influence of LAS on [5-3H]TdR uptake into the soluble nucleotide pool and its incorporation into DNA to detect alterations of cell metabolism not Table 3. Plasma membrane damage and inhibition of growth in V79 cells treated for 4 hr with LAS in HBSS without FCS. Trypan blue staining at time (hr)
100
92 92 88 59 I
2 _ f _ ++ +++ _ +++ +++
Treatment (me/Mm) LAS 3 4 5 5.5 6 6.5 7 1.5
% cell growth
0
3
20
NT
NT
NT
100
+++ +++ +++ +++ +++ +++
+ ++ +++ +++ +++ +++
72 79 44 2 2 4 4 2
NT = not tested The cells were exposed to trypan blue at different times after the end of the treatment. Cell growth was measured 48 hr later from the CD,, nmof the alkali-dissolved monolayers. Number of stained 10%. + + =more than 50%. cells: - =O%, + =about + + + = 100%. All the experiments were run in triplicate.
V. BIANCHIand E. FORTUNATI
12
L---T-J 0.5
1
2
HBSS for OS-2 hr, and immediately afterwards incubated with [5-3H]TdR for 1 hr in complete growth medium. The incubation of untreated cells in HBSS was sufficient by itself to alter TdR uptake: the action of LAS modified it further, decreasing the incorporation of label into the nucleotide pool after 30min. The effect increased with longer exposure times. Inhibition of DNA synthesis was detectable only after 1 hr and did not increase further. In order to test how lasting was the effect of LAS on TdR uptake, cells were treated with LAS for 2 hr and then incubated with [5-3H]TdR either immediately or 1 or 3 hr later. Some representative results are given in Fig. 2: up to 2 mg/litre the effect of LAS on TdR uptake was reversible and limited to the incorporation into the nucleotide pool, while DNA synthesis was similar to that of controls incubated in HBSS. With 4 and 6 mg/litre, DNA synthesis was also markedly and persistently inhibited. On the whole, these experiments indicate that inhibition of DNA synthesis is not one of the primary cytotoxic effects of LAS.
hr Fig. 1. Variations of [5-‘H]TdR uptake into the soluble nucleotide pool (A) and into DNA (B) in cells incubated in HBSS only (A) or HBSS + 2 mg LAS/htre (a) for the indicated times, and then incubated with TdR for 1 hr in
complete growth medium. necessarily dependent on membrane damage. For this purpose we tested LAS concentrations that had proved unable to make the cells permeable to trypan blue. Figure 1 shows the results of one experiment in which the cells were treated with 2 mg LAS/litre in
Release of LDH To look for a specific signal of plasma membrane damage produced by LAS we measured the release of LDH into the treatment solutions. In our hands the sensitivity of the LDH assay was such that at least one million cells had to be exposed to not more than 2 ml of medium in petri dishes of 6 cm diameter. We ran the experiments on cultures of about 1.5 x lo6 cells. In those conditions of cell density, LAS was less effective than in previous experiments on less crowded cultures. Trypan blue staining indicated that membrane damage was less severe than before
.-./ o-o
f
hr
0
$
Fig. 2. Variations of [5-jH]TdR uptake into the soluble nucleotide pool (A, C) and into DNA (B, D) either immediately after 2 hours’ treatment with LAS, or 1 and 3 hr later. All cultures were incubated with TdR for 1hr in complete growth medium. T = beginning of the treatment with: HBSS only (A) or LAS 0.5 (W, 1 0, 2 (01, 4 (Oh 6 (A) mg/htre.
In oitro tests for surfactant toxicity
release of the total LDH activity present in the cells be observed, and it was accompanied by 100% permeability of cells to trypan blue.
4hl
Release l
2hl
I 0
mg/litre
5
7
13
9
Fig. 3. Release of LDH from V79 cells treated with LAS. Cells were treated with S-9 mg LAS/litre in HBSS for 2 (0) or 4 (A) hr and LDH activity was measured in the treatment solution and in the cell layer after solvation with Triton X100. The total LDH content was 0.29 _+ 0.06 U/lo6 control cells (I unit converts I pmol substrate/min) and remained essentially unchanged by the treatments. The activity released is expressed as a percentage of the total activity recovered in each culture & SD in 3 experiments. Where no SD is indicated, data are from 2 experiments only. Parallel cultures were stained with trypan blue. The fraction of stained cells was: - = O%, f. = IO%, + + = more than SO%, + + + = 100%.
of ATP pool components
7
r.--. A
0-O
5
(Fig. 3), probably owing to the reduced cell surface directly exposed to LAS. The influence of cell density on membrane permeability to LAS is confirmed by Fig. 4, where results from different experiments are plotted together. With small deviations, the percentage of cells made permeable decreased, for each dose of LAS, as the number of seeded cells increased. The results on LDH release obtained with treatments of 2 and 4 hr are summarized in Fig. 3: only with 9 mg/litre and 4 hr of treatment could a 30%
and RNA
A more sensitive parameter of membrane damage was the loss of labelled material from cells incubated with [2-3H]Ade before treatment with LAS. The experimental protocol was as follows: after a 1-hr incubation with [Z3H]Ade the cultures were rinsed carefully and exposed to LAS concentrations of 2, 4, or 6 mg/litre in HBSS for l-3 hr; after this time the radioactivity in the incubation medium was measured. Such experiments showed a time- and dose-dependent release of labelled components of the ATP pool from the treated cells (Fig. 5). The total radioactivity in the medium (Fig. 5A) reached its maximum value 1 hr after treatment with 4 and 6 mg LAS/litre; the level was higher with 6 mg/litre, which indicates a more severe damage, not distinguishable by trypan blue staining. About 30% of the radioactivity recovered in the treatment medium was acidprecipitable (Fig. 5B), which suggests leakage of macromolecular nucleic acids. The experimental
10
3 1
.y*
*~.----’
n
100
1
80 v) = 8 $ 60 .E (D 2 40
.
\
Trypan
7mq/litrs
l
Treatment
3
blue
HESS
l
i
&-&-A
I (
\o~Utre
, I 5 10
I 20 seeded
I 30
I 40
A I 50
cellsxlO_'
Fig. 4. Influence of cell density on plasma membrane permeability to LAS. V79 cells were seeded at the indicated inocula in 6-cm petri dishes, and 48 hr later treated with 5-9 mg LAS/like for 2 hr. Membrane permeability was detected by trypan blue staining.
stain1
ng
length of .XPOI”r* 1
\
LAS
20
2 hr
2 mg/litre
-
, Iv,
2
3
-
-
-
+-
4
”
++
+++
+++
6
”
+++
+++
+++
Fig. 5. Release of labelled cell components from V79 cells treated with LAS in HBSS for 1-3 hr immediatelv after 1 hr incubation with [2-3H]Ade. A = total radioactivity recovered in the incubation medium after the treatments. B = acid-precipitable labelled macromolecules released into the medium. HBSS incubation (A), LAS at 2 (a), 4 (O), 6 (A) mg/litre. Parallel cultures were stained with trypan blue, and scored as in Fig. 3.
V. BIANCHIand E. FORTUNATI
14
protocol did not allow exact quantification of the loss of macromolecules because the treatment took place at the end of [2-3H]Ade incubation when the cells were still metabolizing the radioactive precursor. Thus, cells exposed to LAS were releasing nucleotides into the medium while untreated cells were incorporating Iabelled nucleotides into nucleic acids at control rates. Pool and nucleic acid radioactivity in treated cultures was lower than in controls (not shown), but it did not fully reflect the actual losses of material through the ‘holes’ in the membranes, because of the coincidence of two effects: loss of labelled precursors and inhibition of nucleic acid synthesis. To compare directly nucleic acid contents in controls and treated cells, we used another labelhng scheme that also permitted discrimination between partial loss of macromolecular RNA from all (most) cells and complete solubilization of a fraction of the cell population only. According to this second protocol, treatment with LAS was carried out once the utilization of the labelled precursors by cell metabolism had already taken place, and the only expected effect of treatment was the release of cell components into the medium. Preliminary experiments had shown that during a
24 hr chase in unlabelled medium after 24 hours’ incubation with 0.05 pM-[2-3H]Ade, the cells had incorporated 85% of the radioactivity into macromolecular nucleic acids, and the amount of radioactivity in DNA had reached a plateau, with no further incorporation if the chase was prolonged. We treated the cells for 2 hr with LAS (2-6 mg/litre) in HBSS at the end of the 24 hr chase, and then measured the radioactivity in the medium, total nucleic acids and DNA. The results of one such experiment are shown in Fig. 6. The total radioactivity recovered from the medium increased sharply with the dose of LAS, and a fraction, never exceeding 50%, was in precipitable macromolecules (Fig. 6A). The accumulation of radioactivity in the medium was accompanied by a symmetrical decrease of RNA counts in the cells, while the amount of label in the DNA fraction did not change (Fig. 6B). The constant value of the sum of medium plus RNA dpm in the different cultures (Fig. 6C) confirmed that the extracellular radioactivity depended almost exclusively on loss of RNA. This kind of experiment indicated that cell damage produced by LAS was mostly localized in the plasma membrane and in the cytoplasm. DISCUSSION
0
2
4
6
B
n
lrn
c
5
; ,”
3
%
1
.-
.-.-
OLI
t-l---d
mg / litra Fig. 6. Release of labelled cell components from V79 cells treated with LAS for 2 hr after a chase of 24 hr from the end of [2-‘H]Ade incubation. A = radioactivitv recovered in the -incubation medium: total (0). macromolecular (0). B = RNA (0) and DNA (m) .-, radioactivitv in the cells after the treatment. C = sum of the radioactivit; recovered in the incubation medium and in RNA in the different series of cultures.
The plasma membrane is the primary target of surfactants, which perturb its molecular organization and, above a critical level dependent on the nature of the individual surfactant, solubilize membrane components (Helenius and Simons, 1975). The toxicity of surfactants both in vivo and in vitro is strictly related to their ionic properties: a decreasing order of potency for cationic, anionic and nonionic detergents is observed. Cultured cells have been extensively used to study the toxicity of detergents and have consistently produced the toxicity rank mentioned above (Borenfreund and Borrero, 1984; De Leo et al., 1987; Flower et al., 1988; North-Root et al., 1982; Scaife, 1985; Shopsis and Eng, 1985; Shopsis et al., 1985). However, few in vitro data on LAS have been published (De Leo et al., 1987; Pape and Hoppe, 1988) which seems to behave as a typical anionic surfactant. The irritant activity of LAS in human and animal skin is documented (Brown, 1971) and has been attributed to the surfactant-induced release of arachidonic acid from membrane phospholipids (De Leo et al., 1987). We have examined the cellular effects of LAS in vitro in order to provide a basis for the effects detected in aquatic invertebrates in a study on the possible environmental impact of LAS (Bressan et al., 1989). Bressan and co-workers studied the action of LAS, both dissolved in water (a transient situation in the environment) and adsorbed onto sediments (its most frequent and stable condition), on a number of cosmopolitan species of crustaceans, echinoderms, molluscs and oligochaetes. While adsorbed LAS had no effect on any of the species considered, LAS in solution displayed acute effects at concentrations ranging from 0.25 to 200 mg/litre. The induction of lethality by LAS was tested in all species, the impairment of reproductive activity and embryonic development was measured in tubificid oligochaetes and in
In vitro tests for surfactant toxicity
the sea urchin Paracentrotus lividus, and the alteration of some physiological indices (0, consumption, NH, excretion, filtration activity) was monitored in mussels. The highest LD,,s were found with bivalve molluscs, which protected themselves by shutting their valves. The other species were one or two orders of magnitude more sensitive, with copepods and sea-urchin embryos being affected by concentrations below 1 mg/litre. We tried to detect cellular alterations using the range of doses found to be effective in those very sensitive in vivo systems, which gave us the opportunity to compare the sensitivities of some currently used cytotoxicity tests and to devise methods suited to detect fine signals of toxicity. We used general cytotoxicity endpoints (inhibition of cell growth, TdR uptake, trypan blue exclusion) and also more specific indicators of membrane damage (leakage of LDH, nucleotides, RNA). In agreement with a previous report (Benoit et al., 1987) we found that FCS in the medium protects the cells from the cytotoxic action of LAS. The inhibition of cell proliferation caused by a relatively short exposure to LAS in HBSS was abolished by the addition of FCS to the incubation medium. Treatments had to be carried out in HBSS and could last a few hours only, which prevented us from testing the effect of chronic exposures to low concentrations of LAS. This experimental restriction probably accounts for the fact that, even with the most sensitive assays, the doses active in vitro were slightly higher than those active in vivo in copepods and sea-urchin embryos. Trypan blue staining, a simple indicator of cell viability that reflects integrity of membrane structure, gave almost all-or-none answers: very low percentages of cells were stained after treatments with 2-3 mg/litre (about 5-lo%), while more than 50% (and generally 75-80%) of the cells were stained after exposures to 4-5 mg/litre. This sharp increase in cell damage caused by a slight increase in dose is a typical effect of surfactants in cultured cells, not dependent on the parameter of cytotoxicity considered (De Leo et al., 1987; Scaife, 1985; Shopsis and Eng, 1985; Thelestam and Mollby, 1975a and 1976; Walum and Peterson, 1982). In our system 5 mg LAS/litre appeared to be the critical concentration causing the sharp shift in cytotoxicity, but also giving variable results (Tables l-3). Being the threshold dose, 5 mg/ litre was particularly influenced by the experimental variability. Tests based on the incubation of cells with radioactive precursors can reveal a dose- and timedependent enhancement of surfactant cytotoxicity (Thelestam and Miillby, 1976; Walum and Peterson, 1982). That is what we observed with the experiments on (5-3H]TdR uptake (Figs 1 and 2). The incorporation of a labelled precursor in the nucleotide pool, easily assayed by measuring the radioactivity in the soluble cell fraction, is a useful indicator of cytotoxicity, as in the case reported here. It also allows correct evaluation of the inhibition of DNA synthesis due to the treatment (Levis et al., 1978). The protocol followed for the experiment in Fig. 5 indicated that although treatments with 4 and 6mg LAS/litre, made the cells equally permeable to trypan
15
blue, different amounts of soluble pool components were released into the medium. We used adenine rather than uridine (Thelestam and Miillby, 1975a and 1976) to label the cells on the assumption that the ATP pool, being the largest of the nucleotide pools, would give the most sensitive answer in leakage tests. For the same reason other authors have used compounds that are not incorporated into macromolecules, such as a-amino isobutyric acid (Malik et al., 1983; Thelestam and Mollby, 1975b and 1976) 2-deoxy-p-glucose (Walum and Peterson, 1982) or its analogue 2-deoxy-2-fluoro-D-glucose (Malik et al., 1983). While those markers offer the advantage of remaining in the soluble pool fraction instead of being polymerized into macromolecular nucleic acids like the physiological precursors, they compete with the standard components of the growth medium (glucose and amino acids) for transport into the cells, and are easily excreted by the same transport systems that carry them in. As a consequence, the pre-incubation of cells with such radioactive markers has to be carried out in saline (phosphate-buffered saline or HBSS), which by itself alters the normal cell metabolism. Moreover, the duration of treatments is limited by the high spontaneous release of label during the test (Thelestam and Miillby, 1975b). On this basis we preferred to pre-label the cells with a physiological precursor that is not present in the medium (with the exception of the trace amounts contributed by the serum). Adenine is efficiently incorporated into the ATP pool by the salvage enzyme adenine phosphoribosyl transferase and undergoes very low spontaneous release from cells. Our results indicate that by these means it is possible to detect low levels of label release, as with 2 mg LAS/litre for short exposures (Fig. 5A). We have not determined the nature of the low-molecular-weight compounds leaked from the treated cells (Bianchi et al., 1982) and we could not show whether the non-precipitable radioactivity in the treatment media was all in phosphorylated molecules. However, the data obtained by the second protocol of [2-3H]Ade labelling, which clearly indicated loss of macromolecular RNA from cells treated with LAS, also suggested extensive loss of nucleotides. Our [2-3H]Ade labelling protocols effectively detect increasing degrees of membrane damage, and allow discrimination between large ‘functional holes’ produced by detergents and lesser lesions produced by agents such as hexavalent chromium (Fortunati and Bianchi, 1989). In contrast to the sensitivity of the radioactive methods, the LDH leakage test was poorly suited to detect membrane damage. The same observation was reported by Malik et al. (1983). They treated confluent cultures of V79 cells with detergents for 30 min only and found even less LDH release than we did in our conditions. The effect of cell crowding on membrane damage (Fig. 4) can account for those results, and suggests that the LDH test may be useful only with cells in suspension, or presenting high endogenous levels of LDH. A higher LDH release than that observed by us was reported with V79 cells by Lock et al. (1987b) after 17 hours’ exposure to mineral dusts in complete medium containing 10% FCS. As stated above, we could only treat cells
16
V. BIANCHIand E. FORTUNATI
with LAS in serum-free HBSS, which made long treatments inapplicable. On the other hand, some heat-resistant LDH enzyme activity can be present in the serum itself (Benoit et al., 1987), which might interfere with the measurement of low degrees of LDH leakage from the cells treated in complete medium. Acknowledgements-EF was the recipient of a research fellowship from the Antivivisectionist League of Lombardy (LeAL). We are grateful to Professor G. Marin for critically reading the manuscript.
REFERENCES Aeschbacher M., Reinhardt C. A. and Zbinden G. (1986) A rapid cell membrane permeability test using fluorescent dyes and flow cytometry. Cell Viol. Toxicol. 2; 247-255. Benoit J., Cormier M. and Wepierre J. (1987) Effect of proteins on the assessment of surfactan; cytoioxicity by an in vilro test: possible correlations with in vivo data. Toxic. in Vifro 1, 91-96. Bergmeyer H. U. and Bernt E. (1974) UV-assay with pyruvate and NADH. In Methods of Enzymalic Analysis. Vol. 2. pp. 5744578. Academic Press, New York. Bianchi V. and Celotti L. (1985) Accuracy of UV-induced DNA repair in V79 cells with imbalance of deoxynucleotide pools. Mutation Res. 146, 277-284. Bianchi V., Debetto P., Zantedeschi A. and Levis A. G. (1982) Effects of hexavalent chromium on the adenylate pool of hamster fibroblasts. Toxicology 25, 19-30. Bianchi V., Pontis E. and Reichard P. (1987) Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells. Molec. cell. Biol. 7, 42184224. Borenfreund E. and Borrero 0. (1984) In vitro cytotoxicity assays. Potential alternatives to the Draize ocular irritancy test. Cell Biol. Toxicol. 1, 5545. Bressan M., Brunetti R., Campesan G. C., Casellato S., Fava G. C., Giro P., Marin M., Negrisolo P., Tallandini L., Thomann S., Tosoni L. and Turchetto M. (1989) Effect of linear alkylbenzene sulphonate (LAS) on benthic organisms. Tenside, Detergents 26, 148-158. Brown V. K. H. (1971) A comparison of predictive irritation tests with surfactants on human and animal skin. J. Sot. cosmet. Chem. 22, 411420. De Leo V. A., Harber L. C., Kong B. M. and De Salva S. J. (1987) Surfactant-induced alteration of arachidonic acid metabolism of mammalian cells in culture. Proc. Sot. exp. Biol. Med. 184, 477482. Flower C., Phillips B. J. and Anderson D. (1988) Absence of chromosome damage in Chinese hamster ovary cells exposed to detergents. Toxic. in Vitro 2, 65-66.
Fortunati E. and Bianchi V. (1989) Plasma membrane damage detected by nucleic acid leakage. Molec. Toxicol. 1, 27-38. Helenius A. and Simons K. (1975) Solubilization of membranes by detergents. Biochim. biophys. Acta 415, 29-79. Levis A. G., Buttignol M., Bianchi V. and Sponza G. (1978) Effects of potassium dichromate on nucleic acid and protein syntheses and on precursor uptake in BHK fibroblasts. Cancer Res. 38, 110-l 16. Lock S. O., Jones P. A., Friend J. V. and Parish W. E. (1987a) Extracellular release of enzymes from macrophages in vitro for measuring cellular interaction with particulate and non-particulate materials. Toxic. in Vitro i, 77-83. Lock S.O.,Rockley J., Chamberlain M. and Friend J. V. (1987b) Effects of fibrous and non-fibrous dusts on Chinese hamster V79 cells: cytotoxicity or cytostasis? Toxic. in Vitro 1. 71-76. Malik J. K., Schwarz L. R. and Wiebel F. J. (1983) Assessment of membrane damage in continuous cultures of mammalian cells. Chemico-Biol. Interactions 45. 2942. North-Root H., Yackovich F., Demetrulias J., ‘Gacula M., Jr and Heinze J. E. (1982) Evaluation of an in v&-o cell toxicity test using rabbit cornea1 cells to predict the eye irritation potential of surfactants. Toxicology Left. 14, 207-212. Pape W. and Hoppe U. (1988) Estimation of acute irritation potentials of surfactants using our in vivo red blood cell test system. A comparative study. 5th Int. Workshop on In Vitro Toxicology, Schloss Elmau, FRG, November 1988. Scaife M. C. (1985) An in vitro cytotoxicity test to predict the ocular irritation potential of detergents and detergent products. Fd Chem. Toxic. 23, 253-258. Shopsis C., Borenfreund E., Walberg J. and Stark D. M. (1985) A battery of potential alternatives to the Draize test: uridine uptake inhibition, morphological cytotoxicity, macrophage chemotaxis and exfoliative cytology. Fd Chem. Toxic. 23, 259-266. Shopsis C. and Eng B. (1985) Rapid cytotoxicity testing using a semi-automated protein determination on cultured cells. Toxicology Letf. 26, l-8. Thelestam M. and Miillby R. (1975a) Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts. Infect. Immun. 11, 640448. Thelestam M. and Miillby R. (1975b) Sensitive assay for the detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. Infect. Immun. 12, 225-232. Thelestam M. and Miillby R. (1976) Cytotoxic effects on the plasma membrane of human diuloid fibroblasts-A comparative study of leakage tests: Med. Biol. 54, 3949. Walum E. and Peterson A. (19821 Tritiated 2-deoxv-oglucose as a probe for cell mekbrahe permeability studies. Analyl. Biochem. 120, 8-l 1.
Toxic. in Vim Vol. 4, No. I, pp. 9-16, 1990 Printed in Great Britain. All rights reserved
CELLULAR EFFECTS OF AN ANIONIC SURFACTANT DETECTED IN V79 FIBROBLASTS BY DIFFERENT CYTOTOXICITY TESTS V. BIANCHI* Department
and E.
FORTWNATI
of Biology, University of Padua, Via Loredan 10, I-35131 Padova, Italy (Received 10 March 1989; revisions received 12 June 1989)
Abstract-Several cytotoxicity tests were employed to detect the cellular effects of low concentrations of the anionic surfactant linear alkylbenzene sulphonate (LAS). When added to growth medium containing 5% foetal calf serum, LAS did not affect V79 cell growth, nor did it alter the permeability of cell membranes. The inactivity depended on the serum component of the medium. When treatments were carried out in serum-free saline, LAS inhibited cell proliferation, made the plasma membrane permeable to otherwise-undiffusible compounds, and reduced the uptake of tritiated thymidine. The alterations in membrane permeability were evaluated from the release of cytoplasmic molecules of different size (lactate dehydrogenase, adenine nucleotides, RNA) into the medium. The sensitivity of the spectrophotometric lactate dehydrogenase assay was inadequate for the conditions of treatment required to detect the cytotoxicity of LAS. In cultures pre-incubated with tritiated adenine instead, the release of labelled ATP pool components was time and dose dependent and allowed discrimination between levels of membrane damage causing the same degree of trypan blue staining. Also, macromolecular nucleic acids were detected outside the treated cells at doses of 4-6 mg LAS/litre, which indicated severe membrane damage.
INTRODUCTION
At the same time, both the sediments and the organisms can be altered by the detergent. Bressan et al. (1989) examined the acute, subacute and chronic effects of LAS (adsorbed onto the sediment or dissolved in water) on several marine and fluvial benthic species with a wide distribution. Our endpoints for the cellular effects of LAS were general cytotoxicity and specific parameters of membrane damage, and we were interested in detecting effects of LAS doses close to those active
Tissue culture systems have frequently been used as alternatives to in vivo tests in assessing the toxicology of detergents. As components of soaps and shampoos, detergents are generally assayed for their ocular irritation potential by the Draize test on rabbits, but reproducible and predictive in vitro cell systems have been developed for the screening of irritants (Aeschbacher et al., 1986; Borenfreund and Borrero, 1984; De Leo et al., 1987; North-Root et al., 1982; Scaife, 1985; Shopsis and Eng, 1985; Shopsis et al., 1985). Most of the work in vitro has been aimed at obtaining potency ranks comparable with those from the Draize test. We have studied the cytotoxicity of linear alkylbenzene sulphonate (LAS), an anionic surfactant present in domestic detergents, to provide a cellular basis for the in vivo effects detected in a parallel study on benthic invertebrates (Bressan et al., 1989). These authors have investigated the environmental effects of LAS once it reaches surface waters, where it is distributed between water and sediments. The nature of the sediments and the activity of the benthic organisms living on them determine the environmental fate of LAS-adsorption and/or biodegradation.
in vivo.
Several approaches are available for detecting plasma membrane damage, mostly based on variations in membrane permeability. These include the loss of dye exclusion ability (Aeschbacher et al., 1986; Scaife, 1985; Thelestam and Miillby, 1976) leakage of endogenous cell components (Lock et al., 1987a,b; Malik et al., 1983; Scaife, 1985) or the release of radioactive material from cells pre-labelled with physiological precursors or their non-metabolizable analogues (Bianchi et al., 1982; De Leo et al., 1987; Malik et al., 1983; Thelestam and Mollby, 1975a,b; Walum and Peterson, 1982). We tested trypan blue exclusion and lactate dehydrogenase (LDH) leakage, but found them to be rather insensitive indicators of damage. In particular, the LDH leakage test that employs the standard spectrophotometric method to detect LDH activity in the incubation medium appeared to be unsuitable for testing LAS toxicity in our system. A modified protocol, formerly proposed by Thelestam and Miillby (1975a) for measuring nucleotide and RNA release into the medium, allowed us to evaluate the extent of membrane damage from the nature of the released cell components.
*To whom all correspondence should be addressed. Abbreviations: [2-‘H]Ade = tritiated adenine; DMEM = Dulbecco’s modified Eagle’s medium; FCS = foetal calf serum; HBSS = Hanks’ balanced salt solution; LAS = linear alkylbenzene sulphonate; LDH = lactate dehydrogenase; OD,,,, = optical density at 260 nm; (5-3H]TdR = tritiated acid; TCA = trichloroacetic thymidine. 9
10
V. BIANCHI and E. FORTUNATI MATERIALS
AND METHODS
Chemicals. LAS (length of alkyl chain lo-12 carbon atoms, 27.14% in water) was purchased from Italian chemical industries. NADH, pyruvic acid and Triton Xl00 were from Sigma Chemical Co. (St Louis, MO, USA); tritiated thymidine ([5-3H]TdR, sp. act. 25 Ci/mmol) and adenine ([2-3H]Ade, sp. act. 22 Ci/mmol) were from Amersham International (Amersham, England). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from GIBCO BRL (Eggenstein, FRG) and foetal calf serum (FCS) from Seromed, Biochrom KG (Berlin, FRG). All other chemicals and reagents were analytical grade. Cells and culture conditions. The origin and growth characteristics of the V79 line used were those previously described (Bianchi et al., 1987). The cells were routinely grown as monolayers in IO-cm petri dishes at 37’C in a 5% CO, in air atmosphere in DMEM containing 5% heat-inactivated FCS. Cytotoxicity tests. Cells were seeded 24 or 48 hr in advance at a density of lo4 cells/well in 24-well multidishes or at a density of 2 x lo4 tells/3.5-cm dish. For LDH determinations, where more cells were needed, 1.5 x 10’ tells/6-cm dish were plated 48 hr before the experiments. After some preliminary trials (see Results), all treatments with LAS were performed in Tris-buffered Hanks’ balanced salt solution (HBSS), after rinsing the cell monolayers with prewarmed HBSS to remove all traces of FCS. All experiments were run in replicate, and repeated at least twice. On account of the influence of cell density on LAS cytotoxicity (see Results), care was taken to seed the cells always at the same time of the day, and start the treatments accordingly. Experiments with radioactive precursors were run in duplicate, and each point in the graphs is the mean of quadruplicate determinations, with a variability < 10%. Inhibition of cell growth was determined 48 hr after treatment on triplicate cultures: standard deviations were no higher than expected. Cell numbers in control and treated cultures were estimated from their total macromolecular content as follows. The medium was removed from the dishes and the monolayers washed twice with cold HBSS. The soluble nucleotide pool was extracted with 60% methanol for 30 min at 4°C and discarded. The dishes were washed once with 1 ml cold methanol and thoroughly drained before dissolving the cell layer in 0.3 M-NaOH overnight at room temperature. The optical density at 260 nm (GD,bO nm) of the NaOH fraction, which corresponds to the macromolecules left on the plates after pool extraction, is used as an index of cell number (Bianchi and Celotti, 1985). Calibration curves obtained by dissolving in NaOH V79 cultures containing different numbers of cells are linear and indicate that an 0DZ6,,“,,, of 1 corresponds to lo6 cells. To determine the inhibition of [5-‘H]TdR uptake and the level of DNA synthesis in cells treated with LAS, at the end of the treatment the cultures were re-fed with complete medium and incubated for 1 hr with 0.3 pM-[5-3H]TdR either immediately, or 1 or 3 hr later. [5-3H]TdR uptake was measured by counting the radioactivity in the soluble nucleotide pool extracted with 60% methanol as above. Macro-
molecular DNA was obtained by trichloroacetic acid (TCA) precipitation from the 0.3 M-NaOH in which the cell layer was dissolved (Bianchi et al., 1987). Aliquots were collected on glass-fibre filters and counted. The exposure to increasing doses of LAS produced increasing inhibition of [5-3H]TdR uptake into the nucleotide pool. Therefore, original values of DNA radioactivity were normalized on the basis of the percentage variations of radioactivity in the pool (Levis et al., 1978). This allowed a more accurate evaluation of the level of DNA synthesis in the treated cells. Determination of membrane damage. In all experiments trypan blue staining was used as a simple immediate method to monitor the degree of membrane permeability at the end of the treatments. In some cases the cells were stained at different times after treatment to monitor changes of membrane permeability with time. A 0.5% solution of trypan blue was added to an equal volume of the medium and the percentage of stained cells was determined within 5 min by microscopic examination. LDH release was determined by the spectrophotometric procedure of Bergmeyer and Bernt (1974) according to Malik et al. (1983). After treatment the HBSS was centrifuged at 2000 rpm for 5 min and the cell-free supernatant was removed. A 0.5% Triton Xl00 solution in HBSS was used to dissolve the cell layers and cell pellets were obtained by further HBSS centrifugation. The supernatant and the cell lysate were assayed for enzyme activity. Preliminary checks had shown that LAS does not interfere with the activity of standard LDH. Release of ATP pool components. The cultures were incubated for 1 hr with 0.5 PM [2-3H]Ade in complete medium, then repeatedly washed with warm HBSS to remove extracellular radioactivity. The cells were then treated with LAS in HBSS for l-3 hr, the incubation medium was collected, and the plates were processed for pool extraction and cell dissolving as described above. Radioactivity was measured in the medium (total and acid-precipitable), in the nucleotide pool, in total nucleic acids (NaOH fraction) and in DNA (obtained by TCA precipitation of aliquots of the NaOH fraction). Release of macromolecular RNA. To detect loss of RNA from cells exposed to LAS, the cultures were left to settle for 12 hr after seeding and then received 0.05 pM [2-3H]Ade for 24 hr. At the end of incubation the radioactive medium was removed, and plates were rinsed and chased for 24 hr in fresh non-radioactive medium before treating with LAS. After treatment the radioactivity in the treatment solution and in the different cellular fractions was determined as above. With both protocols for [2-3H]Ade incorporation, all the cultures were expected to contain the same amount of tritium, since the incubation with [2-‘H]Ade took place before the treatment with LAS. In order to have a direct check of the effectiveness of the recovery in the different plates, the sum of the radioactivity measured in the medium, the pool and the total nucleic acid fraction of each culture was routinely calculated. A 20% variation in the total radioactivity recovered/culture was the upper limit accepted.
In vitro tests for surfactant toxicity
11
Table 2. Plasma membrane damage in V79 cells treated with LAS in HBSS without FCS
RESULTS
Inhibition of cell growth
Inhibition of cell proliferation in mass culture and reduction of colony-forming ability were considered to be basic indicators of cytotoxicity. A first series of trials, carried out with LAS added to the complete growth medium in a concentration range of 1 pg to 10 mg/litre, showed no effect on cell growth. Plating efficiency in clonal cultures was reduced only above 20mg/litre, with a 50% inhibition at 30 mg/litre and complete cell loss at 50 mg/litre (not shown). The low activity of LAS in complete medium could be attributed to the serum component of the medium, which probably sequestered LAS by its binding to lipoproteins (Benoit et al., 1987). In subsequent experiments the cells were treated in HBSS, where only acute treatments (shorter than a generation time) were possible. The influence of serum was tested by comparing the inhibition of cell growth by LAS in the presence or absence of 5% FCS. Treatments were carried out 24 hr after seeding and cell proliferation was evaluated 48 hr later from the OD,,,, of cell monolayers dissolved in alkali. Table 1 shows the results of two experiments in which V79 cells were treated with 0.5-10 mg LAS/litre in HBSS with or without FCS for 4 hr. Inhibition of growth appeared from 5 mg/litre onwards and was completely abolished by the presence of FCS during treatment. Permeability of plasma membrane
To obtain an indication of cell membrane damage, V79 cells treated with LAS in HBSS without FCS were exposed to trypan blue dye. A semi-quantitative evaluation of membrane damage in cultures treated with 3-7 mg LAS/litre for 1, 2 or 4 hr is given in Table 2. Membrane permeability was monitored immediately after treatment and up to 24 hr later. Effective treatments induced irreversible permeability of cell membranes, with the exception of 7 mg/litre (1 hr) and 5 mg/litre (2 hr), which allowed a partial recovery of membrane integrity. Table I. Influence of FCS on the inhibition of cell growth by LAS dissolved in HBSS with or without FCS* A
Treatment
HBSS - FCS + LAS 0.5 mg/litre I mg/litre 2.5 mg/litre 5 mg/litre 7.5 mg/litre IO mg/litre HBSS + FCS + LAS 0.5 mg/litre I mg/litre 2.5 me/litre _/ 5 mellitre
%,~~
7.5 mg/litre IO mg/litre
Exe.
ODm nm I .70 I .40 NT NT I.36 0.08 NT 0.04 NT NT NT I .62 I .75 NT I.56
1 -A
Em. 2 %t
100
91 6 3 95 103 92
ODmnm I .97 1.61 I .48 I .48 1.41 0.95 0.02 0.02 1.87 1.82 I .88 I .86 1.82 I.81 I .83
%t -
Trypan blue staining after the end of the treatment (hr) LAS treatment (mailitre) I hr
(3) (5) (7)
2hr
(3) (5) (7)
4hr
(3) (5) (7)
0 _ * _ ++ +++ _ +++ +++
100
97 100 99 97 97 98
NT = not tested *V79 cells were incubated for 4 hr in the indicated media 24 hr after seeding. Cell growth was evaluated 48 hr later in triplicate cultures from the OD,,,, of the alkali-dissolved monolayers. iValues are given as % of controls with or without FCS.
4 _
24 _ _
++ +++ +++ +++
_ I!c +++ _ +++ +++
V79 cells were treated with LAS for the indicated times 24 hr after seeding, then exposed to trypan blue at different times after the end of the treatment. Number of stained cells (scored on triplicate plates): - = 0%, f = about IO%, + + = more than 50%. + + + = 100%.
In another experiment (Table 3), a number of doses between 3 and 7.5 mg/litre were tested with 4-hr treatments and the uptake of trypan blue was checked at three time points thereafter. Cell numbers were determined 48 hr after treatment. Again, normal cell permeability was partially restored after 4-5 mg LAS/line, while concentrations higher than 5.5 mg/litre produced irreversible damage and cell detachment, as indicated by the drop in cell density two days after treatment. Tables 2 and 3 show that permeability to trypan blue does not increase progressively: within a very narrow range of doses individual cells change from being impermeable to being fully permeable. Thymidine uptake and DNA synthesis in LAS-treated ceils
The uptake of extracellular TdR and its utilization for DNA synthesis is a complex energy-requiring process that involves many different enzymes. Inhibition of TdR uptake can thus be taken as a broad endpoint of cytotoxicity, being the possible outcome of many individual mechanisms. We examined the influence of LAS on [5-3H]TdR uptake into the soluble nucleotide pool and its incorporation into DNA to detect alterations of cell metabolism not Table 3. Plasma membrane damage and inhibition of growth in V79 cells treated for 4 hr with LAS in HBSS without FCS. Trypan blue staining at time (hr)
100
92 92 88 59 I
2 _ f _ ++ +++ _ +++ +++
Treatment (me/Mm) LAS 3 4 5 5.5 6 6.5 7 1.5
% cell growth
0
3
20
NT
NT
NT
100
+++ +++ +++ +++ +++ +++
+ ++ +++ +++ +++ +++
72 79 44 2 2 4 4 2
NT = not tested The cells were exposed to trypan blue at different times after the end of the treatment. Cell growth was measured 48 hr later from the CD,, nmof the alkali-dissolved monolayers. Number of stained 10%. + + =more than 50%. cells: - =O%, + =about + + + = 100%. All the experiments were run in triplicate.
V. BIANCHIand E. FORTUNATI
12
L---T-J 0.5
1
2
HBSS for OS-2 hr, and immediately afterwards incubated with [5-3H]TdR for 1 hr in complete growth medium. The incubation of untreated cells in HBSS was sufficient by itself to alter TdR uptake: the action of LAS modified it further, decreasing the incorporation of label into the nucleotide pool after 30min. The effect increased with longer exposure times. Inhibition of DNA synthesis was detectable only after 1 hr and did not increase further. In order to test how lasting was the effect of LAS on TdR uptake, cells were treated with LAS for 2 hr and then incubated with [5-3H]TdR either immediately or 1 or 3 hr later. Some representative results are given in Fig. 2: up to 2 mg/litre the effect of LAS on TdR uptake was reversible and limited to the incorporation into the nucleotide pool, while DNA synthesis was similar to that of controls incubated in HBSS. With 4 and 6 mg/litre, DNA synthesis was also markedly and persistently inhibited. On the whole, these experiments indicate that inhibition of DNA synthesis is not one of the primary cytotoxic effects of LAS.
hr Fig. 1. Variations of [5-‘H]TdR uptake into the soluble nucleotide pool (A) and into DNA (B) in cells incubated in HBSS only (A) or HBSS + 2 mg LAS/htre (a) for the indicated times, and then incubated with TdR for 1 hr in
complete growth medium. necessarily dependent on membrane damage. For this purpose we tested LAS concentrations that had proved unable to make the cells permeable to trypan blue. Figure 1 shows the results of one experiment in which the cells were treated with 2 mg LAS/litre in
Release of LDH To look for a specific signal of plasma membrane damage produced by LAS we measured the release of LDH into the treatment solutions. In our hands the sensitivity of the LDH assay was such that at least one million cells had to be exposed to not more than 2 ml of medium in petri dishes of 6 cm diameter. We ran the experiments on cultures of about 1.5 x lo6 cells. In those conditions of cell density, LAS was less effective than in previous experiments on less crowded cultures. Trypan blue staining indicated that membrane damage was less severe than before
.-./ o-o
f
hr
0
$
Fig. 2. Variations of [5-jH]TdR uptake into the soluble nucleotide pool (A, C) and into DNA (B, D) either immediately after 2 hours’ treatment with LAS, or 1 and 3 hr later. All cultures were incubated with TdR for 1hr in complete growth medium. T = beginning of the treatment with: HBSS only (A) or LAS 0.5 (W, 1 0, 2 (01, 4 (Oh 6 (A) mg/htre.
In oitro tests for surfactant toxicity
release of the total LDH activity present in the cells be observed, and it was accompanied by 100% permeability of cells to trypan blue.
4hl
Release l
2hl
I 0
mg/litre
5
7
13
9
Fig. 3. Release of LDH from V79 cells treated with LAS. Cells were treated with S-9 mg LAS/litre in HBSS for 2 (0) or 4 (A) hr and LDH activity was measured in the treatment solution and in the cell layer after solvation with Triton X100. The total LDH content was 0.29 _+ 0.06 U/lo6 control cells (I unit converts I pmol substrate/min) and remained essentially unchanged by the treatments. The activity released is expressed as a percentage of the total activity recovered in each culture & SD in 3 experiments. Where no SD is indicated, data are from 2 experiments only. Parallel cultures were stained with trypan blue. The fraction of stained cells was: - = O%, f. = IO%, + + = more than SO%, + + + = 100%.
of ATP pool components
7
r.--. A
0-O
5
(Fig. 3), probably owing to the reduced cell surface directly exposed to LAS. The influence of cell density on membrane permeability to LAS is confirmed by Fig. 4, where results from different experiments are plotted together. With small deviations, the percentage of cells made permeable decreased, for each dose of LAS, as the number of seeded cells increased. The results on LDH release obtained with treatments of 2 and 4 hr are summarized in Fig. 3: only with 9 mg/litre and 4 hr of treatment could a 30%
and RNA
A more sensitive parameter of membrane damage was the loss of labelled material from cells incubated with [2-3H]Ade before treatment with LAS. The experimental protocol was as follows: after a 1-hr incubation with [Z3H]Ade the cultures were rinsed carefully and exposed to LAS concentrations of 2, 4, or 6 mg/litre in HBSS for l-3 hr; after this time the radioactivity in the incubation medium was measured. Such experiments showed a time- and dose-dependent release of labelled components of the ATP pool from the treated cells (Fig. 5). The total radioactivity in the medium (Fig. 5A) reached its maximum value 1 hr after treatment with 4 and 6 mg LAS/litre; the level was higher with 6 mg/litre, which indicates a more severe damage, not distinguishable by trypan blue staining. About 30% of the radioactivity recovered in the treatment medium was acidprecipitable (Fig. 5B), which suggests leakage of macromolecular nucleic acids. The experimental
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I 30
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Fig. 4. Influence of cell density on plasma membrane permeability to LAS. V79 cells were seeded at the indicated inocula in 6-cm petri dishes, and 48 hr later treated with 5-9 mg LAS/like for 2 hr. Membrane permeability was detected by trypan blue staining.
stain1
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length of .XPOI”r* 1
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-
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Fig. 5. Release of labelled cell components from V79 cells treated with LAS in HBSS for 1-3 hr immediatelv after 1 hr incubation with [2-3H]Ade. A = total radioactivity recovered in the incubation medium after the treatments. B = acid-precipitable labelled macromolecules released into the medium. HBSS incubation (A), LAS at 2 (a), 4 (O), 6 (A) mg/litre. Parallel cultures were stained with trypan blue, and scored as in Fig. 3.
V. BIANCHIand E. FORTUNATI
14
protocol did not allow exact quantification of the loss of macromolecules because the treatment took place at the end of [2-3H]Ade incubation when the cells were still metabolizing the radioactive precursor. Thus, cells exposed to LAS were releasing nucleotides into the medium while untreated cells were incorporating Iabelled nucleotides into nucleic acids at control rates. Pool and nucleic acid radioactivity in treated cultures was lower than in controls (not shown), but it did not fully reflect the actual losses of material through the ‘holes’ in the membranes, because of the coincidence of two effects: loss of labelled precursors and inhibition of nucleic acid synthesis. To compare directly nucleic acid contents in controls and treated cells, we used another labelhng scheme that also permitted discrimination between partial loss of macromolecular RNA from all (most) cells and complete solubilization of a fraction of the cell population only. According to this second protocol, treatment with LAS was carried out once the utilization of the labelled precursors by cell metabolism had already taken place, and the only expected effect of treatment was the release of cell components into the medium. Preliminary experiments had shown that during a
24 hr chase in unlabelled medium after 24 hours’ incubation with 0.05 pM-[2-3H]Ade, the cells had incorporated 85% of the radioactivity into macromolecular nucleic acids, and the amount of radioactivity in DNA had reached a plateau, with no further incorporation if the chase was prolonged. We treated the cells for 2 hr with LAS (2-6 mg/litre) in HBSS at the end of the 24 hr chase, and then measured the radioactivity in the medium, total nucleic acids and DNA. The results of one such experiment are shown in Fig. 6. The total radioactivity recovered from the medium increased sharply with the dose of LAS, and a fraction, never exceeding 50%, was in precipitable macromolecules (Fig. 6A). The accumulation of radioactivity in the medium was accompanied by a symmetrical decrease of RNA counts in the cells, while the amount of label in the DNA fraction did not change (Fig. 6B). The constant value of the sum of medium plus RNA dpm in the different cultures (Fig. 6C) confirmed that the extracellular radioactivity depended almost exclusively on loss of RNA. This kind of experiment indicated that cell damage produced by LAS was mostly localized in the plasma membrane and in the cytoplasm. DISCUSSION
0
2
4
6
B
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3
%
1
.-
.-.-
OLI
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mg / litra Fig. 6. Release of labelled cell components from V79 cells treated with LAS for 2 hr after a chase of 24 hr from the end of [2-‘H]Ade incubation. A = radioactivitv recovered in the -incubation medium: total (0). macromolecular (0). B = RNA (0) and DNA (m) .-, radioactivitv in the cells after the treatment. C = sum of the radioactivit; recovered in the incubation medium and in RNA in the different series of cultures.
The plasma membrane is the primary target of surfactants, which perturb its molecular organization and, above a critical level dependent on the nature of the individual surfactant, solubilize membrane components (Helenius and Simons, 1975). The toxicity of surfactants both in vivo and in vitro is strictly related to their ionic properties: a decreasing order of potency for cationic, anionic and nonionic detergents is observed. Cultured cells have been extensively used to study the toxicity of detergents and have consistently produced the toxicity rank mentioned above (Borenfreund and Borrero, 1984; De Leo et al., 1987; Flower et al., 1988; North-Root et al., 1982; Scaife, 1985; Shopsis and Eng, 1985; Shopsis et al., 1985). However, few in vitro data on LAS have been published (De Leo et al., 1987; Pape and Hoppe, 1988) which seems to behave as a typical anionic surfactant. The irritant activity of LAS in human and animal skin is documented (Brown, 1971) and has been attributed to the surfactant-induced release of arachidonic acid from membrane phospholipids (De Leo et al., 1987). We have examined the cellular effects of LAS in vitro in order to provide a basis for the effects detected in aquatic invertebrates in a study on the possible environmental impact of LAS (Bressan et al., 1989). Bressan and co-workers studied the action of LAS, both dissolved in water (a transient situation in the environment) and adsorbed onto sediments (its most frequent and stable condition), on a number of cosmopolitan species of crustaceans, echinoderms, molluscs and oligochaetes. While adsorbed LAS had no effect on any of the species considered, LAS in solution displayed acute effects at concentrations ranging from 0.25 to 200 mg/litre. The induction of lethality by LAS was tested in all species, the impairment of reproductive activity and embryonic development was measured in tubificid oligochaetes and in
In vitro tests for surfactant toxicity
the sea urchin Paracentrotus lividus, and the alteration of some physiological indices (0, consumption, NH, excretion, filtration activity) was monitored in mussels. The highest LD,,s were found with bivalve molluscs, which protected themselves by shutting their valves. The other species were one or two orders of magnitude more sensitive, with copepods and sea-urchin embryos being affected by concentrations below 1 mg/litre. We tried to detect cellular alterations using the range of doses found to be effective in those very sensitive in vivo systems, which gave us the opportunity to compare the sensitivities of some currently used cytotoxicity tests and to devise methods suited to detect fine signals of toxicity. We used general cytotoxicity endpoints (inhibition of cell growth, TdR uptake, trypan blue exclusion) and also more specific indicators of membrane damage (leakage of LDH, nucleotides, RNA). In agreement with a previous report (Benoit et al., 1987) we found that FCS in the medium protects the cells from the cytotoxic action of LAS. The inhibition of cell proliferation caused by a relatively short exposure to LAS in HBSS was abolished by the addition of FCS to the incubation medium. Treatments had to be carried out in HBSS and could last a few hours only, which prevented us from testing the effect of chronic exposures to low concentrations of LAS. This experimental restriction probably accounts for the fact that, even with the most sensitive assays, the doses active in vitro were slightly higher than those active in vivo in copepods and sea-urchin embryos. Trypan blue staining, a simple indicator of cell viability that reflects integrity of membrane structure, gave almost all-or-none answers: very low percentages of cells were stained after treatments with 2-3 mg/litre (about 5-lo%), while more than 50% (and generally 75-80%) of the cells were stained after exposures to 4-5 mg/litre. This sharp increase in cell damage caused by a slight increase in dose is a typical effect of surfactants in cultured cells, not dependent on the parameter of cytotoxicity considered (De Leo et al., 1987; Scaife, 1985; Shopsis and Eng, 1985; Thelestam and Mollby, 1975a and 1976; Walum and Peterson, 1982). In our system 5 mg LAS/litre appeared to be the critical concentration causing the sharp shift in cytotoxicity, but also giving variable results (Tables l-3). Being the threshold dose, 5 mg/ litre was particularly influenced by the experimental variability. Tests based on the incubation of cells with radioactive precursors can reveal a dose- and timedependent enhancement of surfactant cytotoxicity (Thelestam and Miillby, 1976; Walum and Peterson, 1982). That is what we observed with the experiments on (5-3H]TdR uptake (Figs 1 and 2). The incorporation of a labelled precursor in the nucleotide pool, easily assayed by measuring the radioactivity in the soluble cell fraction, is a useful indicator of cytotoxicity, as in the case reported here. It also allows correct evaluation of the inhibition of DNA synthesis due to the treatment (Levis et al., 1978). The protocol followed for the experiment in Fig. 5 indicated that although treatments with 4 and 6mg LAS/litre, made the cells equally permeable to trypan
15
blue, different amounts of soluble pool components were released into the medium. We used adenine rather than uridine (Thelestam and Miillby, 1975a and 1976) to label the cells on the assumption that the ATP pool, being the largest of the nucleotide pools, would give the most sensitive answer in leakage tests. For the same reason other authors have used compounds that are not incorporated into macromolecules, such as a-amino isobutyric acid (Malik et al., 1983; Thelestam and Mollby, 1975b and 1976) 2-deoxy-p-glucose (Walum and Peterson, 1982) or its analogue 2-deoxy-2-fluoro-D-glucose (Malik et al., 1983). While those markers offer the advantage of remaining in the soluble pool fraction instead of being polymerized into macromolecular nucleic acids like the physiological precursors, they compete with the standard components of the growth medium (glucose and amino acids) for transport into the cells, and are easily excreted by the same transport systems that carry them in. As a consequence, the pre-incubation of cells with such radioactive markers has to be carried out in saline (phosphate-buffered saline or HBSS), which by itself alters the normal cell metabolism. Moreover, the duration of treatments is limited by the high spontaneous release of label during the test (Thelestam and Miillby, 1975b). On this basis we preferred to pre-label the cells with a physiological precursor that is not present in the medium (with the exception of the trace amounts contributed by the serum). Adenine is efficiently incorporated into the ATP pool by the salvage enzyme adenine phosphoribosyl transferase and undergoes very low spontaneous release from cells. Our results indicate that by these means it is possible to detect low levels of label release, as with 2 mg LAS/litre for short exposures (Fig. 5A). We have not determined the nature of the low-molecular-weight compounds leaked from the treated cells (Bianchi et al., 1982) and we could not show whether the non-precipitable radioactivity in the treatment media was all in phosphorylated molecules. However, the data obtained by the second protocol of [2-3H]Ade labelling, which clearly indicated loss of macromolecular RNA from cells treated with LAS, also suggested extensive loss of nucleotides. Our [2-3H]Ade labelling protocols effectively detect increasing degrees of membrane damage, and allow discrimination between large ‘functional holes’ produced by detergents and lesser lesions produced by agents such as hexavalent chromium (Fortunati and Bianchi, 1989). In contrast to the sensitivity of the radioactive methods, the LDH leakage test was poorly suited to detect membrane damage. The same observation was reported by Malik et al. (1983). They treated confluent cultures of V79 cells with detergents for 30 min only and found even less LDH release than we did in our conditions. The effect of cell crowding on membrane damage (Fig. 4) can account for those results, and suggests that the LDH test may be useful only with cells in suspension, or presenting high endogenous levels of LDH. A higher LDH release than that observed by us was reported with V79 cells by Lock et al. (1987b) after 17 hours’ exposure to mineral dusts in complete medium containing 10% FCS. As stated above, we could only treat cells
16
V. BIANCHIand E. FORTUNATI
with LAS in serum-free HBSS, which made long treatments inapplicable. On the other hand, some heat-resistant LDH enzyme activity can be present in the serum itself (Benoit et al., 1987), which might interfere with the measurement of low degrees of LDH leakage from the cells treated in complete medium. Acknowledgements-EF was the recipient of a research fellowship from the Antivivisectionist League of Lombardy (LeAL). We are grateful to Professor G. Marin for critically reading the manuscript.
REFERENCES Aeschbacher M., Reinhardt C. A. and Zbinden G. (1986) A rapid cell membrane permeability test using fluorescent dyes and flow cytometry. Cell Viol. Toxicol. 2; 247-255. Benoit J., Cormier M. and Wepierre J. (1987) Effect of proteins on the assessment of surfactan; cytoioxicity by an in vilro test: possible correlations with in vivo data. Toxic. in Vifro 1, 91-96. Bergmeyer H. U. and Bernt E. (1974) UV-assay with pyruvate and NADH. In Methods of Enzymalic Analysis. Vol. 2. pp. 5744578. Academic Press, New York. Bianchi V. and Celotti L. (1985) Accuracy of UV-induced DNA repair in V79 cells with imbalance of deoxynucleotide pools. Mutation Res. 146, 277-284. Bianchi V., Debetto P., Zantedeschi A. and Levis A. G. (1982) Effects of hexavalent chromium on the adenylate pool of hamster fibroblasts. Toxicology 25, 19-30. Bianchi V., Pontis E. and Reichard P. (1987) Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells. Molec. cell. Biol. 7, 42184224. Borenfreund E. and Borrero 0. (1984) In vitro cytotoxicity assays. Potential alternatives to the Draize ocular irritancy test. Cell Biol. Toxicol. 1, 5545. Bressan M., Brunetti R., Campesan G. C., Casellato S., Fava G. C., Giro P., Marin M., Negrisolo P., Tallandini L., Thomann S., Tosoni L. and Turchetto M. (1989) Effect of linear alkylbenzene sulphonate (LAS) on benthic organisms. Tenside, Detergents 26, 148-158. Brown V. K. H. (1971) A comparison of predictive irritation tests with surfactants on human and animal skin. J. Sot. cosmet. Chem. 22, 411420. De Leo V. A., Harber L. C., Kong B. M. and De Salva S. J. (1987) Surfactant-induced alteration of arachidonic acid metabolism of mammalian cells in culture. Proc. Sot. exp. Biol. Med. 184, 477482. Flower C., Phillips B. J. and Anderson D. (1988) Absence of chromosome damage in Chinese hamster ovary cells exposed to detergents. Toxic. in Vitro 2, 65-66.
Fortunati E. and Bianchi V. (1989) Plasma membrane damage detected by nucleic acid leakage. Molec. Toxicol. 1, 27-38. Helenius A. and Simons K. (1975) Solubilization of membranes by detergents. Biochim. biophys. Acta 415, 29-79. Levis A. G., Buttignol M., Bianchi V. and Sponza G. (1978) Effects of potassium dichromate on nucleic acid and protein syntheses and on precursor uptake in BHK fibroblasts. Cancer Res. 38, 110-l 16. Lock S. O., Jones P. A., Friend J. V. and Parish W. E. (1987a) Extracellular release of enzymes from macrophages in vitro for measuring cellular interaction with particulate and non-particulate materials. Toxic. in Vitro i, 77-83. Lock S.O.,Rockley J., Chamberlain M. and Friend J. V. (1987b) Effects of fibrous and non-fibrous dusts on Chinese hamster V79 cells: cytotoxicity or cytostasis? Toxic. in Vitro 1. 71-76. Malik J. K., Schwarz L. R. and Wiebel F. J. (1983) Assessment of membrane damage in continuous cultures of mammalian cells. Chemico-Biol. Interactions 45. 2942. North-Root H., Yackovich F., Demetrulias J., ‘Gacula M., Jr and Heinze J. E. (1982) Evaluation of an in v&-o cell toxicity test using rabbit cornea1 cells to predict the eye irritation potential of surfactants. Toxicology Left. 14, 207-212. Pape W. and Hoppe U. (1988) Estimation of acute irritation potentials of surfactants using our in vivo red blood cell test system. A comparative study. 5th Int. Workshop on In Vitro Toxicology, Schloss Elmau, FRG, November 1988. Scaife M. C. (1985) An in vitro cytotoxicity test to predict the ocular irritation potential of detergents and detergent products. Fd Chem. Toxic. 23, 253-258. Shopsis C., Borenfreund E., Walberg J. and Stark D. M. (1985) A battery of potential alternatives to the Draize test: uridine uptake inhibition, morphological cytotoxicity, macrophage chemotaxis and exfoliative cytology. Fd Chem. Toxic. 23, 259-266. Shopsis C. and Eng B. (1985) Rapid cytotoxicity testing using a semi-automated protein determination on cultured cells. Toxicology Letf. 26, l-8. Thelestam M. and Miillby R. (1975a) Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts. Infect. Immun. 11, 640448. Thelestam M. and Miillby R. (1975b) Sensitive assay for the detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. Infect. Immun. 12, 225-232. Thelestam M. and Miillby R. (1976) Cytotoxic effects on the plasma membrane of human diuloid fibroblasts-A comparative study of leakage tests: Med. Biol. 54, 3949. Walum E. and Peterson A. (19821 Tritiated 2-deoxv-oglucose as a probe for cell mekbrahe permeability studies. Analyl. Biochem. 120, 8-l 1.