Toxic Effects of Octylphenol on Cultured Rat and Murine Splenocytes

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

139, 437–444 (1996)

0185

Toxic Effects of Octylphenol on Cultured Rat and Murine Splenocytes1 JOYCE U. NAIR-MENON, GARY T. CAMPBELL,

AND

CHARLES A. BLAKE2

Department of Cell Biology and Neuroscience, University of South Carolina, School of Medicine, Columbia, South Carolina 29208 Received January 12, 1996; accepted April 16, 1996

Toxic Effects of Octylphenol on Cultured Rat and Murine Splenocytes. NAIR-MENON, J. U., CAMPBELL, G. T., AND BLAKE, C. A. (1996). Toxicol. Appl. Pharmacol. 139, 437–444. Alkylphenol polyethoxylates and alkylphenols, such as 4-tertoctylphenol (OP), are environmental contaminants. Because these compounds are toxic to aquatic animals, we studied the effects of OP on splenocytes removed from male Fischer 344 rats or male Balb/c mice and cultured in vitro. Cell viability was assessed by trypan blue exclusion after 5 or 27 hr of culture. Culture with 0.08% ETOH (vehicle) or any dose of OP did not alter total cell number or the percentage of viable cells after 5 hr. Culture of cells with two different alkylphenol polyethoxylates for 5 hr resulted in the loss of all cells. The percentages of viable rat or mouse cells after 27 hr of culture were decreased significantly by 10012 M OP or greater concentrations. The actions of OP, dexamethasone (DEX), and 17b-estradiol on rat splenocytes were compared. Dexamethasone was more toxic than OP after 24 hr of culture; 17bestradiol was not toxic. Dexamethasone and OP, but not 17bestradiol, caused significant nuclear condensation after 3 hr of culture (acridine orange staining) or 4 hr of culture (propidium iodide staining). The toxicity of 1006 M OP, but not that of 1006 M DEX, was eliminated when mouse splenocytes were cultured in Ca2/-free medium. Significantly more mouse splenocytes containing free 3*-OH DNA ends were detected by activated cell sorter analyses when the cells had been incubated for 4 hr with 1004 or 1006 M OP or 1006 M DEX. The results of these studies demonstrate that OP is toxic to cultured rat and mouse splenocytes and suggest that this toxic effect is exerted, at least partially, through Ca2/dependent apoptosis. q 1996 Academic Press, Inc.

Alkylphenol polyethoxylates have been used extensively as nonionic surfactants (Etnier, 1986). Because of the largescale use of these compounds it generally is accepted that they are a major determinant of organic material in waste water. Alkylphenol polyethoxylates apparently are degraded aerobically to alkylphenol di- and monoethoxylates which can be further degraded to the respective alkylphenols in anaerobic environments (Giger et al., 1984). 1 Some of this work has been published in abstract form in FASEB Journal 9, A945 (Abstract 5487), 1995 and Molecular Biology of the Cell, Supplement to Vol. 6, 356a (Abstract 2067), 1995. 2 To whom correspondence should be addressed. Fax: (803)733–3212.

The presence of alkylphenol polyethoxylates, alkylphenol di- and monoethoxylates, and alkylphenols in aquatic environments is accepted. However, controversy exists regarding the environmental concentrations of these compounds. The presence of a variety of alkylphenol polyethoxylates in laundry detergents (Marcomini et al., 1988) and of polyethoxylates, alkylphenols, and alkylphenoxy carboxylic acids in effluents from sewage treatment plants and some rivers in Germany and Switzerland (Stephanou and Giger, 1982; Ahel et al., 1987) led to the removal of some of these compounds from some cleaning products. The concentrations of nonionic detergents, predominantly alkylphenol polyethoxylates, in streams and adjacent water wells in Israel have been reported to be in the micromolar range (Zoller, 1993). Alkylphenol polyethoxylates also were detected in Barcelona’s water supply (Rivera et al., 1987). A number of alkylphenol polyethoxylates and derivatives were detected at nanogram per liter concentrations in drinking water in New Jersey (Clark et al., 1992). However, another report suggests that significant environmental contamination of some rivers in the United States by the most commonly used compounds, nonylphenol polyethoxylates and nonylphenol, does not exist (Naylor et al., 1992). The presence of alkylphenol polyethoxylates and derivatives in the environment is troublesome for two reasons. The first concern is the toxicity of these compounds to animal cells. The lethal thresholds for p-nonylphenol for shrimp and salmon ranged from 0.15 to 0.32 mg/liter (McLeese et al., 1981). The compound p-tert-octylphenol was about onethird less toxic to shrimp with a lethal threshold of 1.0 mg/ L (McLeese et al., 1981). A concentration of 140 mg of 2,4di-tert-pentylphenol per gram of fat tissue was found in carp in the Detroit River’s Trenton Channel (Shiraishi et al., 1989). This concentration was slightly higher than the concentration in sediment at the same river site (Shiraishi et al., 1989), indicating that alkylphenols may accumulate in adipose tissue. Thus, alkylphenols at micromolar concentrations, which may be attainable through bioaccumulation, may constitute health hazards to animal cells. Alkylphenols bind to the estrogen receptor and exert estrogenic actions on piscine, avian, and mammalian cells and human breast tumor cells (Mueller and Kim, 1978; Soto et al., 1991; Jobling and Sumpter, 1993; White et al., 1994).

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0041-008X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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The alkylphenol, 4-tert-octylphenol (OP) was the most potent, judging by the ability of 1007 M concentration of OP to significantly displace tritiated 17b-estradiol from its receptor (White et al., 1994). Thus, the second concern is the possibility that OP and related compounds are components of the environmental estrogenic load which has been hypothesized to be responsible for adverse effects on reproductive systems (Sharpe and Skakkebaek, 1993) and a factor in the genesis of human breast tumors (Davis and Bradlow, 1995). For these reasons we elected to study the toxic effects of OP on rat and murine splenocytes. We chose this system for several reasons. First, splenocytes can be obtained and cultured easily. Second, we wanted to partially elucidate the mechanism of the toxicity of OP. To do so, the effects of dexamethasone (DEX) on splenocytes (Schwartzman and Cidlowski, 1993) were used as a model for studying whether OP caused apoptosis. Third, to determine whether the effects of OP on splenocytes were initiated by OP binding to the estrogen receptor, we also used 17b-estradiol in some experiments. MATERIALS AND METHODS Animals. Eight-week-old male Fischer 344 rats and 7-week-old male Balb/c mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA), housed in a room with controlled lighting (12 hr of light, 12 hr of darkness daily) and temperature (20–227C), and used within 4 weeks. Basic cell culture. The preparatory medium was RPMI 1640 (Lot No. 17N5251; Life Technologies, Inc.; Grand Island, NY) containing 2.0% penicillin–streptomycin (Lot No. 23N4152; Life Technologies, Inc.). The culture medium was preparatory medium containing 5.0% fetal bovine serum (Lot No. 91913; Harlan, Indianapolis, IN) and 2 mM glutamine. The animals were decapitated, and the spleens were removed and placed in a sterile dish containing 5 ml of preparatory medium. Splenocytes were forced out of the spleens by gently flushing each spleen with preparatory medium through holes made with a 22-gauge needle. Cells from several spleens were pooled, and the cell suspension was centrifuged. The supernatant was decanted. The red blood cells were lysed by resuspending the pellet in 1.2 ml of 0.16 M ammonium chloride at 377C for 3 min. The lysing procedure was terminated by adding 30 ml of preparatory medium followed by centrifugation. The supernatant was decanted, the pellet was resuspended in 30 ml preparatory medium, and the cell suspension was centrifuged; this procedure was repeated. After this last wash, the cells were resuspended in culture medium. The cell concentration and the viability of the cells were determined by exclusion of trypan blue employing an aliquot of the cells. The volume was adjusted with culture medium so that the cell count was 1 1 107 splenocytes/ml. One-hundred-microliter aliquots of the cell suspension were added to Falcon 12 1 75-mm polypropylene culture tubes (Becton–Dickinson, Lincoln Park, NJ). The alkylphenol, OP (Lot No. JG 14331 EG) and two alkylphenol polyethoxylates, Igepal CA-520 [4-(C8H17)C6H40(CH2CH20)4CH2CH2OH] and Igepal CA-720 [4-(C8H17)C6H40(CH2CH20)11CH2CH2OH] (Lot Nos. JG08422CW and MF-01502DZ, respectively), were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). DEX and 17b-estradiol (Lot Nos. 34H0502 and 120H0126, respectively) were purchased from Sigma Chemical Co. (St. Louis, MO). Solutions of each of the compounds were made in absolute ethanol (ETOH) immediately before the rats or mice were decapitated. Gloves and mask were used as protective clothing while handling these compounds. The final ethanolic solutions were diluted 1:200

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with culture medium. All solutions were made in polypropylene containers. At Time 0 of the culture experiments, 20 ml of the various solutions were added to 100-ml samples of the cell suspensions to give various concentrations of the test agents in culture medium containing 0.08% ETOH. Control cells were cultured in culture medium with or without 0.08% ETOH. Cells were cultured at 377C in a 95% O2 –5% CO2 atmosphere for the desired period of time. In the experiments examining the effects of alteration of extracellular Ca2/ the splenocytes were collected with preparatory medium. After the red blood cells were lysed the remaining cells were washed three times with preparatory medium. After the last wash the cell suspension was divided into two aliquots. The aliquots were centrifuged, and one cell pellet was resuspended in regular culture medium. The other pellet was resuspended in a different culture medium prepared with Ca2/-free RPMI 1640 (Lot No. 18N8347) with 5% fetal bovine serum and 2 mM glutamine added. The cells were washed three times with the respective medium. After the last wash with the respective medium the cell concentrations were determined. The volumes were adjusted with culture medium so that the cell counts were 1 1 107 splenocytes/ml. Cytotoxicity studies. The following numbers of cytotoxicity studies were performed: nine with splenocytes from rats cultured with OP for 27 hr; three with splenocytes from mice cultured with OP or DEX for 27 hr; two with splenocytes from rats and two with splenocytes from mice cultured with OP, DEX, Igepal CA-520, or Igepal CA-720 for 5 hr; 3 with splenocytes from mice cultured with regular culture medium or Ca2/-free RPMI medium containing OP or DEX for 14 hr. In conjunction with three additional experiments with cultures of rat splenocytes that were terminated after 3 hr for acridine orange staining, other samples were allowed to incubate for 27 hr for cytotoxicity studies. In all cytotoxicity studies each agent was tested on duplicate samples. After the appropriate time the cultures were terminated and the percentage cell viability and total cell number were determined by trypan blue exclusion. The values obtained for the duplicate samples were averaged. The average was used for statistical analyses. Acridine orange staining. Rat cells were cultured for 3 hr in regular culture medium containing 0.08% ETOH with or without 1.8 1 1006 M OP, DEX, or 17b-estradiol. Samples (120 ml) of the cell cultures were then stained with acridine orange (10 ml of a solution containing 0.6 mg acridine orange /ml). The cells were not treated with RNase prior to acridine orange staining. The acridine orange (Lot No. 117F-3701) was purchased from Sigma Chemical Co. Cells were visualized with a Zeiss Axiophot fluorescent microscope (Carl Zeiss, Inc., Germany). Photomicrographs were taken of random areas. In one experiment the nuclear areas of 100 cells from each group were determined morphometrically with a Zeiss Videoplan. Propidium iodide staining. Mice splenocytes were incubated for 4 hr with 0.08% ETOH, 1004 or 1006 M OP, or 1006 M DEX. The cell suspensions were centrifuged at 1200 rpm for 10 min, the supernatants were discarded, and the cells were resuspended in 0.5 ml of phosphate-buffered saline (PBS) (pH 7.4). The cells were washed three times with PBS. The final cell pellets were resuspended in 1 ml of cold 1.0% paraformaldehyde in PBS and the cells were fixed on ice for 15 min. The cells were centrifuged and the pellets resuspended in 5 ml of PBS. The cells were washed twice in this manner. After the last centrifugation the cell pellets were resuspended in 0.5 ml 70% ETOH and stored at 0207C until they were stained. The cells were centrifuged and washed three times with PBS and the last pellets were resuspended in 1.0 ml of a 0.1% solution of Triton X-100 in PBS. The cells were vortexed and centrifuged and the Triton X-100 treatment was repeated. The cells were then stained with 1.0 ml of propidium iodide reagent (PBS containing 0.05 mg/ml RNase at 50 U/mg and 5 mg/ ml propidium iodide) for 15 min at room temperature. The propidium iodide (Lot No. 35H3677) was purchased from Sigma Chemical Co. The cells were centrifuged, washed with PBS, and analyzed by flow cytometry. Five thousand propidium-iodide-stained cells were analyzed with a Coulter EPICS V flow cytometer (Coulter Electronics, Inc., Hialeah, FL).

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TOXIC EFFECTS OF OCTYLPHENOL ON MAMMALIAN SPLENOCYTES The samples were excited with an argon laser at 488 nm and emission spectra greater than 630 nm were detected. Cell debris was eliminated from the analyses by elevating forward and 907 side light scatter. ApopTag labeling of 3*-OH DNA ends. The materials were purchased as the ApopTag Apoptosis Detection Kit from Oncor, Inc. (Catalogue No. S7111-KIT; Gaithersburg, MD). The fixed mouse splenocytes in 70% ETOH were centrifuged and washed twice with PBS. The cells were resuspended in 76 ml equilibration buffer. The cells then were centrifuged and the pellets resuspended in 16 ml of working strength terminal deoxynucleotidyl transferase (Tdt) enzyme solution and 38 ml of reaction buffer containing digoxigenin-labeled nucleotides already prepared by Oncor, Inc. They were incubated in a water bath at 377C for 45 min. The cells were vortexed gently every 15 min. This reaction was stopped by centrifuging the cells and resuspending the pellets in 1.0 ml of working strength stop/wash buffer. This step was repeated. The pellets were resuspended in 100 ml of working strength anti-digoxigenin antibody conjugated to fluorescein. The suspension was incubated for 30 min at room temperature with gentle vortexing every 15 min. The cell suspensions were centrifuged, the supernatants removed, and the pellets resuspended in PBS. The cells were washed once more with PBS and the pellets suspended in 1.0 ml PBS for flow cytometry. Five thousand cells were analyzed with a Coulter Profile II flow cytometer. Fluorescein was excited at 488 nm and emission between 510 and 550 nm was detected. Appropriate gating procedures were used to eliminate cell debris. To determine the amount of fluoresence produced by nonspecific binding of the fluorescein-labeled anti-digoxigenin we used cells treated with DEX because we expected DEX to produce internucleosomal cleavage and, thus, substrates for Tdt. However, this sample of cells was not exposed to Tdt. Instead, they were treated with buffer followed by exposure to the antidigoxigenin antibody. This was considered the negative control. Statistics. Data were analyzed with an analysis of variance. The morphometric data were compared with Tukey’s test by the Zeiss Videoplan. We used Duncan’s multiple range test for post-hoc analyses of the rest of the data. p õ 0.05 was considered statistically significant.

RESULTS

The effects of a single exposure of cultured splenocytes from Fischer 344 rats or Balb/c mice to OP are given in Tables 1 and 2, respectively. In both instances, culturing splenocytes in 0.08% ETOH had no effect on the viability.

TABLE 1 The Effects of a Single Exposure to 4-tert-octylphenol (OP) on the Viability of Cultured Fisher 344 Splenocytes Treatment

Percentage of viable cells

Medium 0.08% ETOH 1004 M OP 1008 M OP 10012 M OP 10016 M OP 10020 M OP

76.8 77.7 46.5 60.6 67.1 75.1 74.8

{ { { { { { {

3.0 2.1 3.3* 2.2* 2.7* 2.3 2.6

Total cell No. (1107)/ml 1.01 1.02 1.08 1.00 1.06 1.11 1.07

{ { { { { { {

.06 .08 .09 .07 .08 .08 .05

Note. These data represent the results of nine cytotoxicity studies. The treatments were given at 0 hr and cell viability was determined after 27 hr of culture. Data are expressed as means { standard errors. * Different from the mean 0.08% ETOH value at p õ 0.01.

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TABLE 2 The Effects of a Single Exposure to 4-tert-octylphenol (OP) or Dexamethasone (DEX) on the Viability of Cultured Balb/c Splenocytes Treatment

Percentage of viable cells

Medium 0.08% ETOH 1004 M OP 1008 M OP 10012 M OP 10016 M OP 1006 M DEX

70.0 68.7 21.7 52.7 54.7 61.3 18.3

{ { { { { { {

1.0 0.3 5.4* 0.3* 1.5* 0.9 5.8*

Total cell No. (1107)/ml 1.11 1.02 0.91 1.04 1.07 1.13 0.80

{ { { { { { {

0.21 0.22 0.17 0.20 0.26 0.18 0.17

Note. These data represent the results of three cytotoxicity studies. The treatments were given at 0 hr and cell viability was determined after 27 hr of culture. Data are expressed as means { standard errors. * Different from the mean 0.08% ETOH value at p õ 0.01.

Octylphenol at concentrations of 10012 M or greater significantly decreased the viability of splenocytes. Octylphenol at 1004 M appeared to be slightly more toxic to Balb/c than to Fischer 344 splenocytes. The total number of splenocytes (cells stained and not stained with trypan blue) was not altered by OP or DEX. Similar experiments were conducted in which Fischer 344 or Balb/c splenocytes were cultured with 1006 M concentrations of DEX, OP, Igepal CA-520, or Igepal CA-720 polyethoxylates for 5 hr. Neither DEX nor OP decreased the viability of cells and neither had an effect on total cell number (data not shown). However, the presence of either polyethoxylate for 5 hr resulted in the total loss of all cells (data not illustrated). Comparison of the effects of incubating Balb/c cells in Ca2/-free RPMI with OP or DEX versus incubating them in regular RPMI with OP or DEX for 14 hr is given in Table 3. Incubating cells with 0.08% ETOH vehicle in Ca2/-free RPMI significantly decreased (p õ 0.01) the viability when compared with the viability of cells incubated with 0.08% ETOH vehicle in regular RPMI. With regular RPMI both OP and DEX significantly decreased splenocyte viability. This effect of DEX was greater than that of OP (p õ 0.05). With regular RPMI the percentages of viable cells for OP and DEX were approximately 76 and 53%, respectively, of the percentage of viable cells incubated in the 0.08% ETOH. With Ca2/-free RPMI, DEX, but not OP, significantly decreased splenocyte viability. With Ca2/-free RPMI the percentages of viable cells for OP and DEX were approximately 96 and 74%, respectively, of the percentage of viable cells incubated with Ca2/-free RPMI with 0.08% ETOH. In three experiments Fischer 344 splenocytes were incubated with 1.8 1 1006 M concentrations of OP, DEX, or 17b-estradiol for 3 or 27 hr. The effects of incubation for 3 hr with these agents on nuclear staining with acridine orange

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TABLE 3 The Effects of a Single Exposure to 4-tert-octylphenol (OP) or Dexamethasome (DEX) on the Viability of Balb/c Splenocytes Cultured in Regular RPMI or Ca2/-free RPMI Treatment

Percentage of viable cells

Regular RPMI 0.08% ETOH 1006 M OP 1006 M DEX Ca2/-free RPMI 0.08% ETOH 1006 M OP 1006 M DEX

for 3*-OH DNA ends. In this particular experiment 20% of the splenocytes incubated with 0.08% ETOH had 3*-OH DNA ends as detected with the fluorescein-labeled anti-digoxigenin antibody (Fig. 3b). Octylphenol at 1004 M and DEX at 1006 M increased the percentage of splenocytes with

79.0 { 2.5 60.0 { 5.2* 41.6 { 5.4*,† 55.7 { 1.9 53.7 { 1.3 41.0 { 4.5*

Note. These data represent the results of three cytotoxicity studies. The treatments were given at 0 hr and cell viability was determined after 14 hr of culture. Data are expressed as means { standard errors. The percentage of viable cells cultured with 0.08% ETOH vehicle in Ca2/-free RPMI differed significantly (p õ 0.01) from the percentage of viable cells cultured with 0.08% ETOH vehicle in regular RPMI. * Different from the appropriate mean 0.08% ETOH value at p õ 0.01. † Different from the mean value for cells cultured in regular RPMI with 1006 M OP (p õ 0.05).

are shown in Fig. 1. No evidence of nuclear or chromatin condensation was observed in cells cultured with 17b-estradiol (Fig. 1a), medium (not illustrated), or 0.08% ETOH vehicle (not illustrated). The mean area ({standard error) of nuclei of 100 cells in one experiment cultured with 17bestradiol was 9.1 { 0.22 mm2. In contrast, both DEX and OP led to significant nuclear condensation. The comparable value for the mean nuclear area of 100 DEX-treated cells was 5.7 { 0.11 mm2; the value for 100 OP-treated cells was 5.8 / 0.12 mm2. Dexamethasone and OP also caused chromatin condensations (arrows in Figs. 1b and 1c). The mean percentages of cell viability and standard errors after 27 hr of culture in these three experiments were: medium (80.3 { 1.8), medium with 0.08% ETOH (79.3 {1.2), medium with 1006 M 17b-estradiol (82.2 { 2.0), medium with 1006 M DEX (30.6 { 2.6), and medium with 1006 M OP (61.0 { 1.0). The values for DEX and OP differed from all other values at p õ 0.01. The values for DEX differed from those of OP at p õ 0.01. Flow cytometric analyses disclosed that the intensities of propidium iodide fluoresence in cells incubated with 1.0 1 1004 or 1.0 1 1006 M OP or 1.0 1 1006 M DEX for 4 hr were less than the fluorescence in cells incubated with 0.08% ETOH (Figs. 2a and 2b). The effects of incubating Balb/c cells with 0.08% ETOH, OP, or DEX for 4 hr on the percentages of cells displaying Tdt-extendable 3*-OH DNA blunt ends are illustrated in Figs. 3a–3d. As only 1.0% of cells in the negative control exhibited fluorescence above channel 40 (Fig. 3a), any fluorescence above channel 40 was considered specific staining

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FIG. 1. Cultured rat splenocytes with nuclei stained with acridine orange. Splenocytes were cultured for 3 hr in the presence of 1.8 1 1006 M concentrations of either 17b-estradiol (a), dexamethasone (DEX) (b), or 4tert-octylphenol (OP)(c). The nuclei of cells cultured with DEX or OP are significantly smaller and several examples of chromatin condensation are present (arrows in b and c). The bar represents 10 mM.

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FIG. 2. The frequency of cells exhibiting different intensities of propidium iodide fluoresence (channel numbers) of nuclei. Mouse splenocytes were incubated with 0.08% ETOH (a,b), 1.0 1 1004 (a) or 1.0 1 1006 M octylphenol (OP) (a), or 1.0 1 1006 M dexamethasone (DEX) (b) for 4 hr. The nuclei of cells treated with either dose of OP or with DEX displayed less fluorescence.

3*-OH DNA ends to 43 and 50%, respectively (Figs. 3c and 3d). The means and standard errors for four similar experiments were 0.08% ETOH 24.3 { 1.5%; 1 1 1004 M OP 41.5 { 3.5%; 1 1 1006 M OP 35.8 { 4.8%; 1 1 1006 04 06 M DEX 47.5 { 1.9%. The values for OP at 10 M and 10 06 M and DEX at 10 M were significantly greater (p õ 0.05) than the value for 0.08% ETOH. DISCUSSION

The present results clearly indicate that OP at concentrations of 10012 M or greater is toxic to cultured rat and murine splenocytes. Because the irritating effects of alkylphenols on mammalian skin and eyes are well known (Etnier, 1986), the producers and users of alkylphenols have established safety procedures for personnel likely to come into contact with the alkylphenols. Thus, a primary concern regarding the toxicity of OP to mammalian splenocytes, and other mammalian tissues, is the inadvertent exposure of animals or humans to alkylphenol polyethoxylates and derivatives, including the alkylphenols. A consequent concern would be the amount of these compounds present in the environment. Most reports on the presence of alkylphenols in the environment focus on the presence of nonylphenol. This is because nonylphenol is the alkylphenol from which most of alkylphenol polyethoxylate surfactants are manufactured (Etnier, 1986). Thus, we shall discuss the environmental presence and toxicities of alkylphenol polyethoxylates, alkylphenols, and other derivatives in general and focus on a particular compound when it is appropriate. We think it is reasonable to accept that most of the alkylphenols found in the environment result from degradation of the environmental alkylphenol polyethoxylates. Most environmental alkylphenol polyethoxylates and alkylphenols

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are found in aquatic systems; especially in the inputs to and effluents from waste water treatment facilities and waste water discharges from various industries such as paper mills and textile plants (Stephanou and Giger, 1982; Giger et al., 1984; Rivera et al., 1987; Naylor et al., 1992). Because of the hydrophobic nature of these compounds most of them in aquatic systems are in the sediment phase (Giger et al., 1984; Naylor et al., 1992). However, the concentrations of alkylphenol polyethoxylates in waste water effluents and ground water in Israel have been reported to be in the micromolar range (Zoller, 1993). Additionally, a variety of alkylphenol polyethoxylates and derivatives were found in drinking water in New Jersey at individual concentrations approximating 25 ng/liter (about 10010 M) (Clark et al., 1992). The key concern is whether the concentrations of the alkylphenol polyethoxylates and alkylphenols existing in the environment are high enough to exert deleterious effects on animal cells. For instance, the lethal threshold of nonylphenol on shrimp and salmon is around 1006 M (McLeese et al., 1981). Although 1006 M nonylphenol is greater than the concentrations of nonylphenol reported to date in the aqueous phase of aquatic systems, the lethal threshold gives no information regarding the adverse effects of sublethal concentrations of nonylphenol. The concentrations of alkylphenol polyethoxylates in aquatic systems generally are greater than the concentration of the alkylphenols. Thus, eventual degradation of these compounds may provide a continuing source of environmental alkylphenols. The concentrations of alkylphenols found in the fat of carp from the Trenton Channel of the Detroit River in Michigan (Shiraishi et al., 1989) suggest that alkylphenols may accumulate in animal tissue. Thus, we suggest that the concentrations of alkylphenol polyethoxylates and alkylphenols in some aquatic sys-

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FIG. 3. The frequency of cells exhibiting different intensities of fluorescein fluorescence (channel numbers). Mouse splenocytes were incubated with 0.08% ETOH (b), 1.0 1 1004 M octylphenol (OP)(c), or 1006 M dexamethasone (DEX)(a,d) for 4 hr. Digoxigenin-labeled nucleotides were added to DNA by the activity of terminal deoxynucleotidyl transferase (TDT) (b–d). Digoxigenin was detected by adding an anti-digoxigenin antibody fragment labeled with fluorescein. Terminal deoxynucleotidyl transferase was not added to one batch of cells incubated with 1006 M DEX (a). As only 1.0% of cells shown in (a) exhibited fluorescence above channel 40, fluorescence above channel 40 was considered specific staining for 3*-0H DNA ends. Both OP and DEX increased the number of cells exhibiting staining for 3*-OH DNA ends.

tems is high enough for the compounds to exert adverse biological effects, especially on benthic species. Except for the well-known irritant and toxic effects of alkylphenols on skin and eye tissue (Etnier, 1986) little is known about the possible adverse effects of alkylphenols on other mammalian cells. Two case reports describe the possible association of a nonylphenol polyethoxylated surfactant and free alkylphenols on the presentation of leucoderma (Ikeda et al., 1970) and multiple birth defects (Sherman, 1985). Our observations clearly show that OP at concentrations lower than those observed in some aquatic systems has toxic effects on mammalian splenocytes. These and lower concentrations of OP may have had sublethal effects on significantly more splenocytes. Thus, it appears that, in the absence of information on the bioaccumulation of alkylphenols by mammals, the amount of the precursor alkylphenol polyethoxylates, other derivatives, and alkylphenols in the

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environment should be viewed as a potential health hazard for mammals. With regard to the toxic effects of OP it should be noted that: (a) OP was less toxic than the alkylphenol polyethoxylates we tested and exerted effects through different mechanisms; (b) the toxic effects of OP were not shared by 17bestradiol; and, (c) the toxic effects of OP appeared to be exerted, at least in part, through apoptosis. With regard to the first point, the exposure of splenocytes to 1006 M concentrations of the alkylphenol polyethoxylates for 5 hr resulted in the loss of all cells. As only cell debris was observed in these cultures, it is apparent that these two compounds caused lysis of all cells within 5 hr. The alkylphenol polyethoxylates probably acted as detergents. Exposure of splenocytes to OP for 5 or 27 hr did not result in a significant decrease in the total cell number. Although OP may have been exerting adverse effects on the cell membrane, the

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TOXIC EFFECTS OF OCTYLPHENOL ON MAMMALIAN SPLENOCYTES

integrity of the cell membrane during this time period was sufficient to keep the cells from lysing. The second point revolves around whether OP was acting as an xenoestrogen. Alkylphenols do exert estrogen effects via binding to the estrogen receptor (Mueller and Kim, 1978; Soto et al., 1991; Jobling and Sumpter, 1993; White et al., 1994). Octylphenol concentrations of 1 1 1007 M significantly displaced tritiated 17b-estradiol from the estrogen receptor of the rainbow trout (White et al., 1994). Our splenocyte cell cultures contained mostly lymphocytes. Although it is questionable whether lymphocytes have a significant number of estrogen receptors, 17b-estradiol has not been shown to exhibit toxic effects on splenocyte cell cultures (Forsberg, 1984). Thus, it is most likely that OP was not exerting toxic effects on splenocytes by acting like 17bestradiol. Octylphenol has been reported to exert estrogenic effects on estrogen-responsive tissues at concentrations similar to or greater than those we observed to exert toxic effects on splenocytes. Thus, inadvertent exposure of animals to OP could have adverse effects on mammalian tissues by two mechanisms. The first would be through alkylphenols adding to the xenoestrogen pool (Sharpe and Skakkebaek, 1993; Davis and Bradlow, 1995). The second mechanism would be through the toxic effects. Interestingly, White et al. (1994) reported that OP was not toxic to estrogen-responsive cells at concentrations lower than 1 1 1005 M. An intriguing question is whether estrogen responses provoked by OP rendered these cells resistant to the toxic effects of OP. Third, it is apparent that a short exposure of splenocytes to OP, like a short exposure to DEX, caused several cellular changes which are hallmarks of apoptosis (Wyllie et al., 1980; Darzynkiewicz et al., 1992; Schwartzman and Cidlowski, 1993). As assessed by acridine orange staining, significant nuclear and chromatin condensation were observed in cells exposed to OP. The decreased propidium iodide staining of nuclei of OP- or DEX-treated cells is consistent with early apoptotic changes and may represent loss of low-molecular-weight DNA formed through the action of activated endonuclease from the cell (Darzynkiewicz et al., 1992). Octylphenol and DEX treatment of cells produced specific DNA cleavage so that electrophoresis of the DNA demonstrated the ‘‘ladder’’ pattern of DNA (Nair-Menon, Campbell, and Blake, unpublished observations) consistent with internucleosomal DNA cleavage seen with apoptosis. However, this also was seen in DNA extracted from cells incubated in medium and ETOH. This was not unexpected as some of these cells died during the 27-hr incubation. Thus, we used the Oncor ApopTag in situ Apoptosis detection kit materials for quantitation of DNA cleavage. The materials in this kit detect the amount of cleavage of double-stranded DNA producing extendable 3*-OH DNA blunt ends through the activity of Tdt. Octylphenol and DEX significantly elevated the numbers of cells containing free 3*-0H DNA ends

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available for staining with the ApopTag materials. Incubation of the splenocytes with 0.08% ETOH in Ca2/-free RPMI was itself toxic; however, the additional toxicity of OP was not present in Ca2/-free RPMI. Some additional toxicity of DEX was present with these conditions. Thus, the lymphocytes in the splenocyte cell culture resemble lymphocytes from lymph nodes (Kaiser and Edelman, 1978) and Nb2 lymphoma cells (LaVoie and Witorsch, 1995) rather than thymocytes, as only the latter require extracellular calcium for total DEX-induced apoptosis (McConkey et al., 1989; Kaiser and Edelman, 1978). In summary, our observations suggest that the toxicities of OP and OP polyethoxylates, which are environmental contaminants, should be a concern. Further, at least a portion of the toxic mechanism of OP shares similarities with apoptosis. ACKNOWLEDGMENTS This work was supported by grants from the NIH (DK 38545 and HD 22687). We thank Martha Steele and Neda Osterman for technical assistance and Janice Burns for typing.

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