Effect of a series of triorganotins on the immune function of human natural killer cells

Environmental Toxicology and Pharmacology 23 (2007) 18–24

Effect of a series of triorganotins on the immune function of human natural killer cells Fabiola D. Gomez a , Paula Apodaca a , Laurin N. Holloway b , Keith H. Pannell a , Margaret M. Whalen b,∗ b

a Department of Chemistry, University of Texas at El Paso, El Paso, TX 79968, United States Department of Chemistry, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN 37209, United States

Received 14 February 2006; received in revised form 26 May 2006; accepted 6 June 2006 Available online 10 June 2006

Abstract Natural killer (NK) cells are our initial immune defense against viral infections and cancer development. They are able to destroy tumor and virally infected cells. Thus, agents that are able to interfere with their function increase the risk of cancer and/or infection. Organotins (OTs) have been shown to interfere with the tumor-destroying function of human NK cells. The purpose of the current study was to explore the relationship of a series of triorganotins, that differ in structure by only a single organic group, for their capacity to block NK tumor-cell destroying (lytic) function. Here we examine the series: trimethyltin (TMT), dimethylphenyltin (DMPT), methyldiphenyltin (MDPT), and triphenyltin (TPT). NK cells were exposed to TMT, DMPT, MDPT or TPT for 1, 24, 48 h, or 6 d. A 1 h exposure to TMT, at concentrations as high as 20 ␮M, had no effect on lytic function. However, concentrations as low as 2.5 ␮M were able to decrease NK tumor-destroying function after 6 d. A 1 h exposure to DMPT had no effect on lytic function, however, after 6 d there was an 80–90% decrease in lytic function at 1 ␮M. Exposure to MDPT (as low as 2.5 ␮M) decreased NK function at 1 h, after 6 d there was as much as a 90% decrease at concentrations as low as 100 nM MDPT. TPT decreased lytic function in a manner similar to MDPT, however, it was more effective at 1 h than MDPT. The effect of the triorganotins on the ability of NK cells to bind to targets was studied, to determine if this contributed to the loss of lytic function. The relative immunotoxic potential of this series of compounds is TPT ≈ MDPT > DMPT > TMT. © 2006 Elsevier B.V. All rights reserved. Keywords: Organotins; NK cells; Cytotoxicity; Binding function

1. Intoduction Human natural killer (NK) lymphocytes play a central role in immune defense against virus infection and formation of primary tumors (Lotzova, 1993). NK cells are capable of killing tumor cells, virally infected cells, and antibody coated cells. They are responsible for limiting the spread of blood-borne metastases as well as limiting the development of primary tumors (Kiessling and Haller, 1978; Hanna, 1980). A greatly increased incidence of viral infections has been reported in individuals lacking the NK subset of lymphocytes (Fleisher et al., 1982; Biron et al., 1989). NK cells are defined by the absence of the T-cell receptor/CD3 complex, the presence of CD56 and/or

∗

Corresponding author. Tel.: +1 615 963 5247; fax: +1 615 963 5326. E-mail address: [email protected] (M.M. Whalen).

1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.06.001

CD16 on the cell surface, and the presence of cytoplasmic granules containing cytolytic proteins such as granzyme B and perforin (Lotzova, 1993). Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. Organotin (OT) are used in a variety of industrial and agricultural applications (Laughlin and Linden, 1985; Kannan and Lee, 1996; Federal Register, 2000; USDA, 2000, 2001). OT contamination has been reported in water, sediment and fish from both freshwater and marine environments in the United States, Europe and Japan (Alzieu et al., 1991; Fent and Hunn, 1991; Kannan et al., 1995a,b,c; Tolosa et al., 1992; Ueno et al., 1999; Borghi and Porte, 2002). Environmental contamination resulting from extensive use of OTs has been of great concern due to the potential for deleterious effects on non-target organisms (Kimbrough, 1976; Kannan et al., 1997; Maguire, 2000; Nielsen and Strand, 2002).

F.D. Gomez et al. / Environmental Toxicology and Pharmacology 23 (2007) 18–24

Organotins include methyltins (MTs), butyltins (BTs) and phenyltins (PTs). Tributyltin (TBT) has been used in wood preservation, marine antifouling paints, disinfection of circulating industrial cooling waters, and slime control in paper mills (Kimbrough, 1976; Roper, 1992). Detectable levels of TBT have been found in fish (Kannan et al., 1995a,b,c) and various household products such as siliconized-paper baking parchments (Yamada et al., 1993). Measurable levels of TBT have been found in human blood (Kannan et al., 1999; Whalen et al., 1999). The human intake of TBT was estimated to be 2.29 ␮g/d in a market basket survey done in Japan in 1997 (Sekizawa, 1998). Triphenyltin (TPT) is an organotin, which is approved for use as a fungicide on major food and food-stock crops, including sugar beets, potatoes, pecans, and rice (Kannan and Lee, 1996; Federal Register, 2000; USDA, 2000, 2001). Human exposure to PTs might come from occupational exposure and/or consumption of contaminated food (Kimbrough, 1976; Borghi and Porte, 2002). Rats fed TPT showed diminished thymus dependent immune responses, such as delayed-type hypersensitivity reactions (Snoeij et al., 1987). Triphenyltin hydroxide produced tumors in rats and mice (Roper, 1992; Snoeij et al., 1987). Previously, we have shown that both TBT and TPT are very potent inhibitors of human NK cells’ ability to destroy tumor cells (Whalen et al., 1999, 2000; Wilson et al., 2004). Thus, these compounds have the capacity to increase the risk of cancer development by blocking this important immune defense against tumor cells. Trimethyltin (TMT) is present in some PVC products at levels of 8.5–24.9 ␮g/g (Hiroyuki et al., 2002). TMT appears to be primarily neurotoxic (Brown et al., 1979; Jenkins and Barone, 2004; Ross et al., 1981). There have been documented cases of accidental acute exposures to this compound (Fortemps et al., 1978; Gui-bin et al., 2000). Additionally, measurable levels of TMT have been found in urine samples from humans who had not experienced an acute exposure (Braman and Tompkins, 1979). There are to our knowledge no studies examining the immunotoxic effects of TMT on human NK cells. A study in rats indicated that TMT induced atrophy of spleen, thymus, and lymph nodes (Hioe and Jones, 1984). However, this same study found no effect on NK cell activity. Separate studies indicated that TBT and TPT were much more immunotoxic in rats than TMT (Snoeij et al., 1985, 1986). The current study will examine the effects of TMT on the immune function of human NK cells. Additionally, we will examine the effects the series of OTs: TMT, dimethylphenyltin (DMPT), methyldiphenyltin (MDPT) and TPT on the tumordestroying function of human NK cells. This study will allow for the first time a comparison of the immunotoxicity of OTs as they progress from one triorganotin species (TMT) to another (TPT). TMT is much less hydrophobic than TPT (Jensen et al., 1991). TMT may also have other chemical characteristics (such as capacity to interact with biomolecules) that differ from those of TPT (Nath et al., 2001). This type of study has not previously been possible due to the lack of availability of heteroleptic organotins. DMPT and MDPT

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are not commercially available and were synthesized for these studies. It is important to determine the relationship between toxicity to NK function and the structure of the triorganotins in determining the mechanism of triorganotin immunotoxicity. Here we compare TMT, DMPT, MDPT, and TPT for their ability to interfere with the ability of human NK cells to destroy tumor cells. 2. Materials and methods 2.1. Isolation of NK cells Blood samples (buffy coats) were obtained from American Red Cross (ARC), Portland, OR, which obtained informed consent. We were approved for receipt and use of blood products by the Institutional Review Board (IRB) of the ARC as well as by the IRB of our university. Highly purified NK cells were obtained using a rosetting procedure. Buffy coats were mixed with 1 mL of RosetteSepTM human NK cell enrichment antibody cocktail (StemCell Technologies, Vancouver, British Columbia, Canada) per 30 mL of buffy coat. The mixture was incubated for 40 min at room temperature with periodic mixing. Following the incubation 4 mL of the mixture was layered onto 4 mL of Ficoll-Hypaque (1.077 g/mL) (Sigma), and centrifuged at 1200 × g for 20 min. The cell layer was collected and washed twice with phosphate buffered saline (PBS; 10 mM phosphate (pH 7.2)/2.7 mM KCl/140 mM NaCl) and stored in complete media (RPMI-1640 supplemented with 10% heat-activated BCS, 2 mM l-glutamine and 50 U penicillin G with 50 ␮g streptomycin/mL) at 1 million cells/mL. The resulting cell preparation was >95% CD16+ , CD56+ 0% CD3+ by fluorescence microscopy and flow cytometry (Whalen and Loganathan, 2001; Whalen et al., 2002).

2.2. Chemical preparation TMT and TPT were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO). DMPT and MDPT were synthesized as previously described (Apodaca et al., 2001; Kapoor et al., 2005). Stock solutions of each compound were made in dimethylsulfoxide (DMSO) (Sigma–Aldrich). Stock solutions of the compounds were diluted in gelatin media (0.5% gelatin replaced the calf serum in complete media) to achieve the desired concentrations. The final concentration of DMSO did not exceed 0.01%.

2.3. Cell treatments Purified NK cells were separated by centrifugation from complete media (defined above) and transferred to complete media containing 0.5% gelatin in place of the 10% bovine serum. This was done in order to avoid binding of the hydrophobic compounds to serum albumin, which could interfere with their delivery to the cells. NK cells were then exposed to the compounds for 1, 24, 48 h or 6 d. The concentration ranges examined for each of the compounds were: TMT, 20 ␮M–500 nM; DMPT, 10 ␮M–100 nM; MDPT, 5 ␮M–25 nM; and TPT, 1 ␮M–25 nM.

2.4. Cell viability Cell viability was determined by trypan blue exclusion. Cell numbers and viability were assessed at the beginning and end of each exposure period. The viability of treated cells was compared to that of control cells at each concentration and length of exposure (Whalen et al., 2003).

2.5. Chromium release assay NK cytotoxicity was measured using a 51 Cr release assay (Whalen, 1997; Whalen et al., 1999). The target cell in all cytotoxicity assays was the NKsusceptible K562 (human chronic myelogenous leukemia) cell line. K562 cells were incubated with 51 Cr in 0.2–0.5 mL of BCS for 1 h at 37 ◦ C in air/CO2 (19:1). Following this incubation the target cells were washed twice with gelatin

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medium. NK cells (1.2 × 105 /100 ␮L for 12:1 ratio with target cells) were added to the wells of round-bottomed microwell plates. The NK were diluted to 6:1 ratio (0.6 × 105 /100 ␮L) and 3:1 ratio (0.3 × 105 /100 ␮L); each ratio was tested in triplicate. Target cells were added (1 × 104 /100 ␮L) to each well, and the plate was centrifuged at 300 × g for 3.5 min and incubated for 2 h at 37 ◦ C (air/CO2 , 19:1). After incubation a 0.1 ml aliquot of the supernatant was collected and counted for radioactivity for 60 s in a Packard COBRA gamma radiation counter (Packard Instrument Co., Meriden, CT). Target lysis was calculated as follows: 100 × [(test c.p.m.−spontaneous c.p.m.)/(maximum c.p.m.−spontaneous c.p.m.)]. Maximum release was produced by adding 100 ␮L of target cells to 100 ␮L of 10% Triton X-100. Statistical analysis of the data was carried out utilizing ANOVA and Student’s t-test. Data were initially compared within a given experimental setup by ANOVA. A significant ANOVA was followed by pair wise analysis of control versus exposed data using Student’s t-test.

Table 1 Viability of lymphocytes following exposure to TMT, DMPT, MDPT, and TPT Compound

3. Results

Percentage of viable cells as compared with control at each length of exposure 24 h

48 h

6d

TMT

10 ␮M 5 ␮M 2.5 ␮M 1 ␮M 0.5 ␮M 0.25 ␮M

101 94 98 93 99 100

± ± ± ± ± ±

4 4 9 8 3 8

89 102 103 108 103 94

± ± ± ± ± ±

30 16 19 12 26 20

77 87 101 100 105 112

± ± ± ± ± ±

22a 23 10 19 21 8

DMPT

10 ␮M 5 ␮M 2.5 ␮M 1 ␮M 0.2 ␮M 0.1 ␮M

93 104 101 100 98 95

± ± ± ± ± ±

10 7 15 16 14 3

81 98 101 98 98 99

± ± ± ± ± ±

5a 11 11 8 11 9

67 77 97 93 94 97

± ± ± ± ± ±

36a 25a 14 11 19 20

MDPT

1 ␮M 200 nM 100 nM 50 nM 25 nM 10 nM

83 107 106 109 110 104

± ± ± ± ± ±

23 10 4 3 2 7

45 76 102 92 102 94

± ± ± ± ± ±

35a 36 10 8 8 8

72 68 88 97 95 80

± ± ± ± ± ±

16a 17a 22 16 22 28

TPT

200 nM 100 nM 50 nM 25 nM

99 99 102 93

± ± ± ±

7 7 9 7

98 97 103 107

± ± ± ±

15 14 8 9

99 98 89 97

± ± ± ±

22 16 4 12

2.6. Conjugation assay The percentage of target cells with bound NK cells was determined at two effector to target ratios, 12:1 and 6:1. The NK cells were treated as described above for the cytotoxicity assay. Tumor cells were added to an appropriate number of control or treated NK cells in a round-bottomed microwell plate (20,000 tumor cells/well). The plate was centrifuged at 300 × g for 3.5 min. Each condition was tested in triplicate. The cells were incubated at 37 ◦ C, air/CO2 , 19:1, for 10 min and then placed on ice. The cells were then gently resuspended with a micropipet, placed in a hemocytometer, and viewed under a light microscope. The number of target cells with two or more NK cells bound to their surface was counted, as well as the total number of targets to determine the percentage of tumor cells with NK cells bound. The minimum number of targets counted per determination was 50–100 (Whalen et al., 1993).

Concentration

a A significant decrease in viability as compared to control. There was 100% recovery of cells following exposures.

3.1. Viability of NK cells exposed to varying concentrations of OTs

3.3. Cytotoxic function of purified NK cells exposed to DMPT for 1, 24, 48 h, and 6 d

Table 1 shows the effects of exposures to varying concentrations of TMT, DMPT, MDPT and TPT for varying lengths of time on the viability of NK cells as compared to control cells. Only those concentrations of compound that did not significantly decrease viability (after a given length of exposure 24, 48 h, and 6 d) were studied for effects on the ability of NK cells to lyse tumor cells.

Exposure of NK cells to DMPT for 1 h caused no significant decreases (p > 0.05) in cytotoxic function at any concentration (10–1 ␮M) (Fig. 2). A 24 h exposure to 10 ␮M DMPT caused a 98 ± 3% decrease in the capacity of NK cells to lyse tumor cells (Fig. 2, p < 0.0001). Exposure to 2.5 ␮M DMPT for 24 h was able to decrease NK lytic function by 52 ± 16% (p < 0.0001). Exposure to 5 and 2.5 ␮M DMPT for 48 h decreased NK lytic function

3.2. Cytotoxic function of purified NK cells exposed to TMT for 1, 24, 48 h, and 6 d Highly purified NK cells were exposed to TMT: 20–5 ␮M for 1 h; 10–0.5 ␮M for 24 h; 10–0.5 ␮M for 48 h; and 5–0.5 ␮M for 6 d (Fig. 1). Following the exposure period the TBT-containing medium was removed and the cells were assayed for tumor-cell lysing (cytotoxic) function using the 51 Cr release assay. Exposure of NK cells to TMT for 1 h caused no significant decreases (p > 0.05) in cytotoxic function at any concentration. After 24 h, 10 ␮M caused a very significant decrease (76 ± 18%) in the capacity of NK cells to lyse tumor cells (p < 0.0001). A 48 h exposure decreased the ability of NK cells to lyse tumor cells at concentrations as low as 2.5 ␮M (23 ± 12%, p < 0.001). A 6 d exposure to 5 ␮M TMT caused a very significant loss of cytotoxic function (96 ± 4%, p < 0.0001). Exposure to 2.5 ␮M for 6 d also decreased NK function (49 ± 11%, p < 0.0001).

Fig. 1. Effect of exposures to TMT on the cytotoxic function of human NK cells: 1 h exposure to 5–20 ␮M TMT; 24 h exposure to 0.5–10 ␮M TMT; 48 h exposure to 0.5–10 ␮M TMT; 6 d exposure to 0.5–5 ␮M TMT. Data were combined from replicate experiments using cells from different donors. To combine results from separate experiments the levels of lysis were normalized as percentage of the lytic function of the control cells in a given experiment. Results were the average ± S.D. of three determinations for 1 h; and 9 or more determinations for all other time points.

F.D. Gomez et al. / Environmental Toxicology and Pharmacology 23 (2007) 18–24

Fig. 2. Effect of exposures to DMPT on the cytotoxic function of human NK cells: 1 h exposure to 1–10 ␮M DMPT; 24 h exposure to 200 nM to 10 ␮M DMPT; 48 h exposure to 100 nM to 5 ␮M DMPT; 6 d exposure to 100 nM to 2.5 ␮M DMPT. Data were combined as described in Fig. 1.

by 91 ± 1% and 71 ± 4%, respectively (Fig. 2, p < 0.0001). A 48 h exposure significantly decreased the ability of NK cells to lyse tumor cells at concentrations as low as 1 ␮M (45 ± 9%, p < 0.0001) (Fig. 2). A 6 d exposure to 1 ␮M DMPT decreased lytic function by 84 ± 14% (p < 0.0001) (Fig. 2). 3.4. Cytotoxic function of purified NK cells exposed to MDPT for 1, 24, 48 h, and 6 d In contrast to TMT and DMPT, exposure of NK cells to MDPT for 1 h caused very significant decreases in NK lytic function at both 5 and 2.5 ␮M (93 ± 7% and 66 ± 5%, respectively, p < 0.0001) (Fig. 3). Exposure to 200 and 100 nM MDPT for 24 h caused 83 ± 13% and 62 ± 27% decreases, respectively, in the capacity of NK cells to lyse tumor cells (Fig. 3, p < 0.0001 and p < 0.01). The effects of a 48 h exposure to MDPT were similar to those seen at 24 h. A 6 d exposure to 100 nM MDPT decreased lytic function by 84 ± 12% (p < 0.0001) and 50 nM MDPT decreased function by 37 ± 18% (p < 0.01, Fig. 3). 3.5. Cytotoxic function of purified NK cells exposed to TPT for 1, 24, 48 h, and 6 d Exposure of NK cells for 1 h to 1 ␮M, 500 and 200 nM TPT caused decreases in NK lytic function of 95 ± 3%, 84 ± 7%, and 31 ± 9% (p < 0.0001, Fig. 4). Exposure to 200 nM TPT for 24 h

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Fig. 4. Effect of exposures to TPT on the cytotoxic function of human NK cells: 1 h exposure to 100 nM to 1 ␮M TPT; 24 h exposure to 25–200 nM TPT; 48 h exposure to 25–200 nM TPT; 6 d exposure to 25–200 nM TPT. Data were combined as described in Fig. 1. n = 9 for 1, 24 and 48 h; n = 12 for 6 d.

caused an 86 ± 11% decrease in the capacity of NK cells to lyse tumor cells (Fig. 4, p < 0.0001). After a 48 h exposure to 200 nM TPT lysis was inhibited by 94 ± 5% (p < 0.0001, Fig. 4). A 6 d exposure to TPT caused a decrease in lytic function of 38 ± 27% at 50 nM (p < 0.001, Fig. 4). 3.6. Binding function of NK cells exposed to TMT The effect of TMT exposure to alter the ability of NK cells to bind to tumor target cells was examined at those concentrations of TMT that were able to decrease cytotoxic function. NK cells exposed between 10 and 5 ␮M TMT for 24 h showed no decrease in their ability to bind K562 cells (data not shown). There was also no effect on binding after a 48 h exposure to TMT. However, after a 6 d exposure to TMT there was a greater than 90% decrease in binding function at 5 ␮M (p < 0.02) (Fig. 5A) 3.7. Binding function of NK cells exposed to DMPT Exposures of NK cells to 10, 5, or 2.5 ␮M DMPT for 24 h caused no change in binding function (data not shown). Likewise, 48 h exposures caused no decrease in binding function at 5 or 2.5 ␮M DMPT (data not shown). A 6 d exposure to DMPT caused significant decreases in binding function (greater than 80%) at the 2.5 ␮M concentration (p < 0.01) (Fig. 5B). 3.8. Binding function of NK cells exposed to MDPT The ability of NK cells to bind to K562 tumor cells was unaffected by exposure to MDPT for 24 h. The concentrations tested were 200, 100, and 50 nM. Exposure to these same concentrations of MDPT for 48 h also caused no significant change in the ability of NK cells to bind to tumor cells (data not shown). However, a 6 d exposure to 100 nM MDPT decreased the binding capacity of NK cells by about 40% at the 6:1 ratio (p < 0.01) (Fig. 5C). 3.9. Binding function of NK cells exposed to TPT

Fig. 3. Effect of exposures to MDPT on the cytotoxic function of human NK cells: 1 h exposure to 200 nM to 5 ␮M MDPT; 24 h exposure to 25–200 nM MDPT; 48 h exposure to 25–200 nM MDPT; 6 d exposure to 25–100 nM MDPT. Data were combined as described in Fig. 1. n = 6 for 1, 24 h, and 6 d; n = 9 for 48 h.

NK cells exposed to TPT for 24 h showed no significant change in their ability to bind to tumor cells. However, after a 48 h exposure to 200 nM TPT binding of NK cells to tumor cells

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F.D. Gomez et al. / Environmental Toxicology and Pharmacology 23 (2007) 18–24

Fig. 5. Effects of exposures to TMT, DMPT, MDPT, and TPT on the ability of NK cells to bind to K562 tumor target cells: (A) 6 d exposure to 1–5 ␮M TMT; (B) 6 d exposure to 0.2–2.5 ␮M DMPT; (C) 6 d exposure to 50–100 nM MDPT; (D) 48 h exposure to 50–200 nM TPT; (E) 6 d exposure to 25–200 nM TPT. Results were the average ± S.D. of 3–9 determinations.

was decreased by about 40% (p < 0.01) (Fig. 5D). Lower concentrations of TPT had no significant affect on binding at 48 h. A 6 d exposure to 200 nM TPT caused a significant decrease in binding function (70–80%, p < 0.01) (Fig. 5E). NK cells exposed to 100 nM TPT for 6 d also exhibited decreased binding function (greater than 50%, p < 0.01) (Fig. 5E). 4. Discussion The current study examined the effects of TMT, DMPT, MDPT, and TPT on the tumor-cell destroying function of human NK cells. This study provides for the first time a comparison of the immunotoxic effects of OTs as they progress from one homoleptic OT species (TMT), through the heteroleptic compounds (DMPT and MDPT) to another homoleptic OT (TPT). When a given OT compound was shown to decrease the ability of NK cells to lyse tumor cells, we went on to examine whether this loss of function might be due to a loss of the capacity of the NK cell to bind to the tumor target. When NK cells were exposed to TMT for 1 h there was no alteration of their capacity to destroy tumor cells, even at concentrations as high as 20 ␮M. However, with increasing length of exposure there were measurable decreases in this function at

concentrations as low as 2.5 ␮M (Fig. 1). Anytime a loss of lytic function is observed in viable NK cells, it is important to assess whether this loss is due to a loss of their ability to bind to the target. Thus, any concentration of compound that was effective at decreasing lysis was evaluated for its effect on the capacity of the NK cell to bind to the tumor cell using a conjugation assay. Binding studies indicated that there was no decrease in the capacity of the NK cells to bind to tumor cells when the NK cells were exposed to TMT for 24 or 48 h. Thus, the losses of lytic function seen with TMT exposures of 24 or 48 h could not be accounted for by decreased ability of the NK cell to bind to its target. There was a greater than 90% loss of binding function after a 6 d exposure to 5 ␮M TMT (Fig. 5A). Thus, loss of binding may explain the loss of lytic function seen at this concentration and length of exposure to TMT. The replacement of one methyl group with a phenyl group caused an increase in the capacity of the OT to diminish NK function. For instance, a 24 h exposure to 2.5 ␮M DMPT caused an approximately 50% decrease in lytic function (Fig. 2). However, this same concentration of TMT had no effect on NK function at 24 h or even after 48 h (Fig. 1). DMPT had no effect on the ability of NK cells to bind K562 tumor targets after 24 or 48 h at any concentration, but 2.5 ␮M decreased binding by greater

F.D. Gomez et al. / Environmental Toxicology and Pharmacology 23 (2007) 18–24

than 80% after 6 d. Thus, as with TMT loss of binding function could not explain the loss of lytic function seen after 24 or 48 h exposures to DMPT. The loss of lytic function seen at 2.5 ␮M after 6 d could be largely accounted for by decreased binding. However, the very significant loss of lytic function seen after a 6 d exposure to 1 ␮M DMPT could not be accounted for by the effects of that concentration of DMPT on binding. Replacement of two of the methyl groups with phenyl groups had a very significant impact on the capacity of the compound to interfere with NK cell function. As seen in Table 1, 1 ␮M MDPT began to decrease the viability of the NK cells after a 24 h exposure (and thus this concentration was not studied beyond 1 h). NK cells exposed to 2.5 ␮M MDPT for 1 h showed a very significant loss (66%) of lytic function (Fig. 3). Neither TMT nor DMPT were able to decrease lytic function to any extent following a 1 h exposure to any concentration tested. While 200 nM MDPT was able to decrease lytic function after 24 h by about 80%, this same exposure had no effect on the capacity of the NK cells to bind to tumor cells. This result indicates that the loss of ability of the NK cell to destroy the tumor cell (at 24 and 48 h) was not due to an inability to bind to the target. This was similar to what was seen with TMT and DMPT. When all three organic groups in the OT were phenyl groups the compound became more effective at decreasing NK lytic function following a 1 h exposure (Fig. 4). However, TPT’s ability to decrease lysis after 24 h, 48 h, and 6 d exposures was similar to that of MDPT. Exposure to 200 nM TPT for 48 h decreased the capacity of the NK cells to bind to tumor cells by about 40%. Thus, the loss of binding could explain in part the loss of lytic function seen at this exposure. However, the loss of lytic function was about 90% under this condition so it appears that there may be other aspects to the loss of function. Previously, we have shown that TBT is a very potent inhibitor of NK lytic function (Whalen et al., 1999; Wilson et al., 2004). TBT can decrease lytic function at concentrations as low as 10 nM in some donors. In comparison to TBT, none of the compounds tested in this study were as potent at decreasing lytic function. However, each of the tested compounds has some ability to diminish function with both MDPT and TPT approaching the effectiveness of TBT. Thus, these compounds may have the capacity to increase the risk of cancer development by blocking this important immune defense against tumor cells. There have been no previous studies of the effects of TMT on human NK function. As measurable levels of TMT have been found in urine samples from humans who had not experienced an acute exposure (Braman and Tompkins, 1979) it was important to determine its potential immunotoxicity in humans. A study in rats indicated that TMT had no effect on NK cell activity (Hioe and Jones, 1984). Other studies indicated that TBT and TPT were much more immunotoxic in rats than TMT (Snoeij et al., 1985, 1986). In the current study we demonstrated that TMT decreased human NK function within 24 h at the 10 and 5 ␮M levels. TMT has previously been shown to decrease neurite length and neuronal cell line viability at concentrations of 4 ␮M and above (Jenkins and Barone, 2004). Thus, the levels of TMT where we observed effects on human NK function were simi-

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Fig. 6. Comparison of the effects of TMT, DMPT, MDPT, and TPT on NK cytotoxic function. Data are presented as percent of control function. One hour compares the effect of 5 ␮M of each OT (except TPT which is 1 ␮M); 24, 48 h, and 6 d compare the effects of 200 nM exposures to each OT.

lar to those where neurotoxicity was seen (Jenkins and Barone, 2004). TMT is much less lipophilic than TPT. TMT has a measured log Po/w of −1.2, while that of TPT is 1.5 (Jensen et al., 1991). Increased lipohilicity would allow the compound to cross the cell membrane more readily. Once in the interior of the cell the compound could interfere with molecular processes required for NK lytic function (Aluoch and Whalen, 2005). The fact the most lipophilic compound tested, TPT, was also the most rapid and effective (significant loss of function at 200 nM within 1 h) in its inhibitory effects (Fig. 4) is consistent with the suggestion that access to the cell interior (and specific molecular components) is essential to the loss of function. Thus, the increase in capacity to block NK lytic function as the OTs progressed from TMT to TPT may at least in part be due the increase in the lipophilic nature of the compounds. This study provides important information regarding the immunotoxicity of TMT and the relationship between OT structure and immunotoxicity. In summary the results of this study indicated that: (1) TMT has the ability to decrease the lytic function of human NK cells; (2) the ability of the OTs to diminish the lytic function of human NK cells increased in the following sequence, TMT, DMPT, MDPT, TPT (Fig. 6); (3) the decrease in lytic function seen with any of the OTs could not be entirely explained by a loss of NK capacity to bind to the target cell; (4) the increase in the lipophilic nature of the OTs appears to increase their immunotoxicity. Acknowledgments This research was supported by Grant 2S06GM-0809228 (MMW), Grant GM08012 (KHP) and MARC program GM008048 (KHP) from the National Institutes of Health. References Aluoch, A., Whalen, M.M., 2005. Tributyltin-induced effects on MAP kinases p38 and p44/42 in human natural killer cells. Toxicology 209, 263–277. Alzieu, C., Michel, P., Tolosa, I., Bacci, E., Mee, L.D., Readman, J.W., 1991. Organotin compounds in Mediterranean: a continuing cause for concern. Mar. Environ. Res. 32, 261–270.

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