Sharnise Wilson, * Bommanna G. Loganathan, and Margaret M. Whalen Department of Chemistry, Tennessee State University, Nashville, TN 37209 *Department of Chemistry, Murray State University, Murray, KY 42071.
Introduction
Organotin compounds are used as heat stabilizers polyvinyl chloride polymers, industrial and agricultural biocides and as industrial catalysts in variety of chemical reactions [1]. Triphenyltin (TPT) is an organotin, which is primarily used in agriculture as a contact fungicide to treat wide variety of crops including potatoes, peanuts, carrots, soybeans, sugar beets and rice [2-4]. TPT is also used in antifouling paints to prevent growth of barnacles and other fouling organisms on aluminum hulled boats and ships over 75 ft. Due to its extensive use, TPT contamination has been reported in water, sediment and fish from both freshwater and marine environments in the United States, Europe and Japan [5-8]. Laboratory TPT exposure studies have revealed a wide spectrum of toxic effects in aquatic and terrestrial organisms. Immune function studies with rats fed TPT revealed that the thymus dependent immune responses were suppressed including delayed-type hypersensitivity reactions. Triphenyltin hydroxide produced tumors in rats and mice [4,9].
Human exposure to phenyltins (PTs) might come from occupational exposure and/or consumption of contaminated food. Very limited information is available on the toxic effects of TPT in humans. Because of the deleterious effects of this compound, the Occupational Safety and Health Administration (OSHA) has established workplace exposure limits of 0.1 mg m3 of air for organotin compounds [4]. The Food and Agricultural Organization of the United Nations/ World Health Organization's (FAO/WHO) proposed a provisional tolerable daily intake (TDI) of 0.5 痢 TPT/kg body wt/day [10]. Human NK cells play a central role in immune defense against virus infection and formation of primary tumors [11]. Previously, we have shown that exposure to TPT (1 然 for 1 h) greatly reduced the ability of NK cells (obtained from peripheral blood of adult male and female volunteer donors) to kill K562 (human chronic myelogenous leukemia) tumor cells [12]. The ability of NK cells to lyse tumor targets was measured using a 51Cr release assay [12]. In this study we examined whether the inhibition of NK-cell cytotoxicity induced by a brief (1 h) exposure to TPT could be reversed if the TPT was removed and the cells were allowed to incubate for up to 6 days in TPT-free media.
Material and Methods
Blood Sampling and Isolation of NK Cells:
Peripheral blood samples from healthy adult (male and female), volunteer donors
were used for this study. Whole blood was applied to Ficoll-Hypaque (1.077 g/ml)
and centrifuged at 500 g for 30 min. to remove red blood cells. Mononuclear
cells were collected from the Ficoll-Hypaque and washed twice (250 g, 10 min.)
with phosphate buffered saline (PBS). In order to remove platelets, the cells
were suspended in a small volume (2.5 - 5 ml) of PBS and layered on to 2.5-5 ml
of bovine calf serum (BCS) and centrifuged at 200 g for 5 min. The pellet was
resuspended in PBS and the process was repeated. The cells were then suspended
in complete medium which consisted of RPMI-1640 supplemented with 10%
heat-activated BCS, 2 mM
L-glutamine and 50 U penicillin G with 50 痢 streptomycin/ml. Non adherent
cells (30-40% CD16+, 60-70% CD3+ ) were prepared by incubating the cells in
glass Petri dishes (150 X 15 mm) at 37 蚓 and 5% CO2 for 1 h.[12-14].
Chemical Preparation:
TPT was purchased from Aldrich Chemical Co. WI, USA. Dimethylsulfoxide (DMSO)
was obtained from Sigma Chemical Co. St. Louis, MO, USA). A Neat standard was
resuspended in DMSO. TPT was diluted in gelatin media (0.5% gelatin replaced the
calf serum in complete medium) to achieve a final concentration of 750 nM (the
final concentration of DMSO did not exceed 0.0005%.
Cell Viability and Cytotoxicity:
Cell viability was determined by trypan blue exclusion. Prior to assay, the NK
cells were separated by centrifugation from complete medium (RPMI 1640) and
transferred to gelatin media. Cell numbers and viability were assessed at the
beginning and end of the assays. Cell numbers and their viability did not vary
among experimental conditions. NK cytotoxicity was measured using a 51Cr release assay [12-14]. The target
cell in all cytotoxicity assays was the NK-susceptible K562 (human chronic
myelogenous leukemia) cell line. An appropriate number of K562 cells were
incubated with 51Cr in 1 ml of BCS for 1.5 hr at 37
蚓 in air/CO2 (19:1). The cells were then washed twice with gelatin medium. NK
(effector) cells (5.0x105/100 無 for 50:1 ratio) were added to the wells of
round-bottom microtitre plates. The effectors were diluted to 25:1 ratio (2.5x105/100
無), and 12.5:1 ratio (1.25x105/100 無); each ratio was tested in
triplicate. Targets were added (1x104/100無) to each well, and the
plate was centrifuged at 150 g for 3 min. and incubated for 2 hr at 37 蚓 (
air/CO2 ,19:1). After incubation 0.1 ml aliquot of the supernatant
was collected and counted for radioactivity for 60 sec in a Packard COBRA gamma
counter (Packard Instrument Co., Meriden, CT, USA) gamma radiation counter.
Specific lysis was calculated as follows: 100x[(test c.p.m - spontaneous
c.p.m.)/maximum c.p.m.- spontaneous c.p.m.)]. Maximum release was produced by
adding 100 無 of 10% Triton X-100.
Results and Discussion
Lymphocytes were exposed to 750 nM TPT for 1h. The concentration of TPT used in treating the cells was a concentration that reduced cytotoxic function by between 50-70% after a 1 h exposure, while not affecting cell viability. Following the exposure period, the TPT containing media was removed and replaced with TPT-free media. The cells remained in TPT free media for varying lengths of time (0, 24 h, 48 h, 4 days and 6 days) and were assayed for cytotoxic function using a 51Cr release assay.
Figure 1 shows the percent inhibition of NK cytotoxicity when NK cells were exposed to 750 nM TPT for 1h followed by a 0 h, 24 h, 48 h, 4 d or 6 d period in TPT-free media at the 50:1 lymphocyte: target ratio. When the cells were assayed for cytotoxic function immediately after a 1 h exposure to 750 nM TPT ( 0 h recovery) there was a 57+10% decrease in NK-cytotoxic function at the 50:1 ratio, (n=12). When TPT-exposed NK cells were allowed to incubate in TPT-free media for 24 h (24 h recovery period), NK-cytotoxic function was decreased by 84+15% at the 50:1 ratio (n=12). Specific lysis was decreased by 83+25% following a 48 h incubation in TPT-free media (n=9, 50:1 ratio). A 1 h exposure to 750 nM TPT followed by a 4 d incubation in TPT-free media produced an 86+19% inhibition of cytotoxicity (n=6, 50:1 ratio). After a 6 day incubation period in TPT-free media cytotoxicity was reduced by 98+4% at the 50:1 ratio (n=6). Thus, there was no significant recovery of cytotoxic function even when the NK cells were allowed to incubate in TPT-free media for up to 6 days. There was a significant difference in percent inhibition of cytotoxicity between the 0 h recovery time and all other recovery time points (p<0.001) when a pair wise comparison was carried out using Student's t test. That is, there was a greater decrease in cytotoxic function in the cells that were given a recovery period than in those that were given no recovery period (0 h in TPT-free media). This indicated that exposure to TPT induced changes in the NK cells that lead to an increased inhibition of function even in the absence of compound in the media. This indicated that TPT was acting as an irreversible inhibitor of cytotoxic function whose effect(s) increased with time.
Triphenyltin hydroxide appeared to have an effect on immunity in both rats and mice as shown by the moderate changes in immunoglobulins IgG, IgA, IgM in both males and females [4]. An accidental exposure of phenyltin lead to death of a female worker reported following renal failure 12 days after exposure [15]. Human exposure to TPT can occur by multiple routes (occupational exposure during the manufacture, application in the agricultural fields, consumption of contaminate fish from coastal as well as freshwater ecosystems etc.). Since human exposure to phenytins is possible as evidenced by the presence of these compounds in fishery products [20] and there are demonstrated negative effects of these compound on NK cell function in as little as one hour. There is a need for further studies dealing with the mechanism by which TPT disrupts essential immune functions such as NK cytotoxicity.
References
1. Kimbrough, R.D. (1976). Environ. Health Perspect. 14, 51-56.
2. Kannan, K. and Lee, R.F. (1996). Environ. Toxicol. Chem. 15,1492-1499.
3. Kannan, K., Senthilkumar, K., Loganathan, B.G., Takahashi, S., Odell, D.K. and Tanabe, S. (1997). Environ. Sci. Technol. 31, 296-301.
4. Roper, W.L. (1992). Toxicological profile for tin. U.S. Department of Health and Human Services. Agency for Toxic Substances and Disease Registry, U.S.A.
5. Alzieu, C., Michel, P., Tolosa, I., Bacci, E., Mee, L.D. and Readman, J.W. (1991). Marine Environ. Res. 32, 261-270.
6. Fent, K. and Hunn, J. (1991). Environ. Sci. Technol. 25, 956-963.
7. Tolosa, I., Mertini, L., de Bertrand, N., Bayona, J.M. and Albaiges, J. (1992). Environ. Toxicol. Chem. 11,145-155.
8. Kannan, K., Tanabe, S. and Tatsukawa, R. (1995). Chemosphere 30,925-932.
9. Snoeij, N.J., Penninks, A.H. and Seinen, W. (1987). Environ. Research 44, 335-353.
10. FAO/World Health Organization Monographs (1971). Evaluation of some pesticide residues in food.
11. Trinchieri, G. (1989). Adv. Immunol. 47, 187-376.
12. Whalen, M.M., Hariharan, S. and Loganathan, B.G. (2000). Environ. Research 84, 162-169.
13. Whalen, M.M., Loganathan, B.G. and Kannan, K. (1999). Environ. Research. 81, 108-116.
14. Whalen, M.M., and Loganathan, B.G. (2001) Toxicol. Appl. Pharmacol. 171, 141-148.
15. NIOSH. (1976). Criteria for a recommended standard -occupational exposure to organotin compounds. Report to National Institute for Occupational Safety and Health, Cincinnati, OH. NIOSH-77-115. No. 274766.
16. Ueno, S., Susa, N., Furukawa, Y., Komatsu, Y., Koyama, S. and Suzuki, T. (1999). Arch. Environ. Health 54, 20-25.
|
If you have come to this page from an outside location click here to get back to mindfully.org |