* Aquatic Toxicology Laboratory, Department of Zoology, National Food Safety and Toxicology Center and Institute of Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824;
Department of Pediatrics and Human Development and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824; and
Reproductive Toxicology Division, National Health and Environmental Effects Laboratory, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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Key Words: GJIC; PFOS; perfluorinated chemicals; rodents; QSAR.
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INTRODUCTION |
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Perfluorooctane sulfonic acid (PFOS) appears to be the ultimate degradation product of a number of perfluorinated compounds used in commercial applications (Giesy and Kannan, 2001). The concentrations of PFOS found in wildlife are greater than other perfluorinated compounds (Giesy and Kannan, 2001
; Kannan et al., 2001a
,b
). However, to date, most toxicological studies have been conducted using perfluorinated fatty acids, such as perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA), rather than the more environmentally prevalent sulfonated compounds. Whether PFOS can cause similar effects as PFOA and other PFFAs is still under investigation, and its possible mechanism(s) of action remains to be elucidated.
Gap junctions are plaque-like features on the cell plasma membrane formed by connexin proteins (Yamasaki et al., 1995). Each connexin protein is composed of 6 subunits, forming a pipeline-like structure with a center pore of about 17 Å in diameter (Yeager and Nicholson, 1996
). When these protein complexes from adjacent cells join, they form a continuous channel structure, and allow electronic and metabolic signaling molecules to pass through the channel to symchronize tissue function, a process called gap junctional intercellular communication (GJIC; (Bruzzone et al., 1996
).
Of the various forms of intercellular connection, GJIC is the only one that allows direct exchange of chemicals from the interior of one cell to that of adjacent cells without passage through the extracellular space (Pitts and Finbow, 1986). The cytosolic molecules that can be exchanged through GJIC include ions, second messengers, and low molecular weight metabolites (Yamasaki, 1996
). GJIC is considered to play an essential role in maintaining the homeostasis of tissues, therefore disruption of GJIC results in abnormal cell growth and function (Trosko et al., 1998
). Because tumor formation requires loss of homeostasis and abolition of contact inhibition, it has been hypothesized that the inhibition of GJIC is associated with tumor promotion (Trosko and Ruch, 1998
). A recently developed quantitative structure activity relationship (QSAR) model has demonstrated that inhibition of GJIC is strongly linked to tumor development in rodents, uncontrolled cell proliferation and differentiation, embryonic lethality or teratogenesis (Ketcham and Klaunig, 1996
). Chronic disruption of GJIC could also lead to neurological, cardiovascular, reproductive, and endocrinological dysfunction (Trosko et al., 1998
).
Previous studies have shown that PFFAs with carbon chain lengths of 710 can rapidly and reversibly inhibit GJIC in a dose-dependent fashion in vitro (Upham et al., 1998). To compare the effects of the sulfonic acid class of PFFAs with various chain lengths on GJIC to those of other PFFAs, and to evaluate possible species and organ differences, the inhibition of GJIC was studied in the WB F-344 rat liver cell line and the CDK dolphin kidney cell line treated with PFOS and related perfluorinated compounds. The dolphin cell line was used here in an effort to develop a marine mammalian model for testing the effect of PFOS since relatively great concentrations of PFOS have been measured in marine mammal tissue samples, particularly liver samples (Kannan et al., 2001b
). In addition, an in vivo study with subchronic exposure of Sprague-Dawley rat to PFOS was conducted to determine whether effects on GJIC observed in vitro might be relevant in vivo.
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MATERIALS AND METHODS |
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Cell culture.
Rat liver epithelial cells (WB-F344) were obtained from J. W. Grisham and M. S. Tsao, University of North Carolina. This cell line has been well characterized for its expression of gap junction proteins (Ruch and Trosko, 2001) and oval cell characteristics (Tsao et al., 1984
). Carvan dolphin kidney (CDK) cell line was obtained from D. Busbee, Texas A & M University. The CDK line is an epithelial cell line isolated from a prematurely born female-bottle-nose dolphin (Carvan et al., 1994
). WB-F344 and CDK cells were cultured in 75 cm2 flasks (Corning 430720) in a humidified incubator at 37°C, with a 5/95% CO2/air atmosphere. WB-F344 cells were cultured in Dulbeccos Modified Eagle Medium (Formula 78-5470EF, Gibco, Rockville, MD), supplemented with 5% Fetal Bovine Serum (FBS; Gibco, Rockville, MD). CDK cells were cultured in Dulbeccos Modified Eagle Medium and Hams F12 medium (Sigma, St. Louis, MO), supplemented with 10% FBS (Gibco, Rockville, MD), and other nutrients (Carvan et al., 1994
).
Animals and treatment.
Sixty-day-old Sprague-Dawley rats (males 294 ± 4 g; females 209 ± 2 g) were obtained from Charles River Laboratories (Raleigh, NC), and housed at 2024°C and humidity-controlled (4060%) facilities at the U.S. EPA Reproductive Toxicology Division. Rats were randomly assigned to either block 1 with 6 males and 6 females or block 2 with 4 males and 4 females. Block 1 was exposed to PFOS for 21 days, block 2 was exposed for 3 days. Within each block, half of the males and half of the females were randomly assigned to treatment or control groups. Rats received PFOS (5 mg/kg) or vehicle control (0.5% Tween-20) daily by gavage at a rate of 1 ml/kg body weight. Food and water were provided ad libitum.
GJIC in vitro assay.
After reaching 90100% confluence, cells were harvested with 1x trypsin-EDTA (Gibco, Rockville, MD) and the resulting cell suspension was diluted to approximately 1 x 106 cells/ml for WB-F344 cells, and 1 x 105 cells/ml for CDK cells. Two-ml aliquots of the diluted cell suspension were transferred to 35 mm diameter tissue culture plates, and cells were incubated for approximately 72 h until confluence was reached. Test compounds, dissolved in acetonitrile, were added to culture medium to assess effects on GJIC. Doses used and exposure durations are discussed in the results section for each experiment.
GJIC in vitro was measured using the scrape loading dye transfer technique (Weis et al., 1998). Briefly, following the exposure to compounds of interest, the cells were washed 3 times with phosphate buffered saline (PBS). The fluorescent dye, lucifer yellow (Sigma, St. Louis, MO) dissolved in PBS (1 mg/ml) was applied to cover the cells. Three parallel scrapes were made in the cell monolayer using a surgical blade to allow passage of the membrane impermeable dye into ruptured cells. After a 3-min incubation, the cells were washed with PBS to remove excess dye and were fixed with 4% formalin. Dye migration was observed and photographed at 200X using a Nikon epifluorescence microscope illuminated with an Osram HBO 200W lamp and equipped with a COHU video camera. The program, Gel-Expert (Nucleotech, San Mateo, CA), was used to quantify GJIC by determining the intensity and distance of dye migration. The distance of dye migration perpendicular to the scrape (i.e., between adjacent cells linked only by gap junctions) represents the ability of cells to communicate via GJIC. Dye migration data are reported as a percentage of the corresponding mean control value. All treatments were tested in triplicate. NOEL and EC50 values were determined by one-way ANOVA and linear regression analyses. Differences among compounds and between cell types were determined using two-way ANOVA, followed by Tukeys multiple range test.
GJIC in vivo assay.
GJIC activity after in vivo exposure was measured using the incision loading/dye transfer technique (Krutovskikh et al., 1991; Sai et al., 2000
). At the end of exposure period, rats were sacrificed by decapitation, the left lobe of the liver was excised immediately and rinsed with PBS. Lucifer Yellow (1 mg/ml in PBS) was applied onto the tissue surface. Four incisions (
1 cm long, 1 mm deep) were made on each of the tissue samples with a surgical blade. Additional dye solution was loaded into the incisions with a pipette tip, and the specimen was incubated for 5 min at room temperature. After incubation, the specimen was washed 3 times with PBS, and fixed in 10% buffered formalin overnight. Specimens were trimmed, mounted in tissue processing cassettes, and paraffin embedded. Sections, 5 µm, were prepared by cutting the paraffin block perpendicular to the incision lines on the liver specimen. Dye migration was quantitated using the same optical and data processing systems used for the in vitro assay. Three incisions were analyzed for each specimen; results were analyzed using nested ANOVA. Samples of each liver were also collected, and stored at 80°C for chemical analysis.
Chemical analysis.
PFOS in the rat liver tissue samples was extracted and analyzed based on slight modifications of previously described methods (Hansen et al., 2001). Extractions were carried out on homogenate volumes equivalent to 1050 mg of the original liver tissue samples. Homogenates, prepared in nanopure water, were mixed with an equal volume of 0.5 M tetrabutylammonium (TBA) hydrogen sulfate, pH 10 and 0.25 M sodium carbonate buffer. After mixing, the sample was extracted twice with methyl-tert-butyl ether (MTBE). The MTBE was evaporated to dryness and the extract was resuspended in 1 ml methanol for transfer to injection vials. After transfer, methanol was removed by evaporation and the extract was resuspended in 200 µl of 50% methanol in 2 mM ammonium acetate. PFOS was analyzed using a Hewlett Packard 1100 HPLC system (Hewlett Packard, Palo Alto, CA) interfaced to a Micromass Platform II mass spectrometer (Micromass, Beverly, MD). Chromatography was conducted on a 150 x 4 mm Betasil C18 column (Keystone Scientific, Bellefonte, PA). Concentrations were calculated based on a standard curve generated with at least 5 PFOS concentrations that were run 3 times at the start, middle, and end of the analytical run. All calculations and curve fitting were performed with MassLynx software (Micromass, Beverly, MD).
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RESULTS |
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In Vivo Results
Exposure of rats to PFOS for 3 days did not alter the animals body weight gain; in contrast, significant deficits were seen after 21 days of treatment (Table 2). Although low, measurable levels of PFOS were detected in control rat liver, after 3 days of treatment, about 125 µg PFOS/g liver was detected; after 21 days of treatment an average of 725 µg PFOS/g liver was measured (Table 3
). The low concentrations of PFOS measured in control rats are presumably due to sources of contamination associated with the rats before purchase. GJIC was significantly reduced in liver tissue from PFOS-treated rats after 3 days of exposure (Figs. 1EF
, Fig. 4
). The magnitude of inhibition was the same for the extended exposure up to 21 days. Since only a single dose concentration of PFOS was assessed it was not possible to develop a dose-response relationship for the in vivo exposure. No significant difference was detected between males and female rats in either the control or treatment groups.
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DISCUSSION |
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The regulation of GJIC occurs at different levels of cellular control (Yamasaki et al., 1995) including mutation of connexin genes, reduced connexin gene expression, increased mRNA degradation, altered connexin protein translational control, posttranslational phosphorylation as well as binding of chemicals to the connexin proteins. The connexin molecules that constitute gap junctions are produced in the golgi apparatus. Therefore, the translocation of connexin from the golgi apparatus to cell membrane could also be a point of modulation of GJIC activity. Previous studies have shown that the peroxisome proliferator activated receptor (PPAR) mediates many of the effects of peroxisome proliferators, including perfluorinated compounds (Issemann and Green, 1990
). Whether this receptor is potentially involved in this process is still under investigation; however, to date there is no direct evidence of a relationship between GJIC inhibition and PPAR.
While the mechanism of GJIC inhibition by perfluorinated compounds is not fully understood, the results of these experiments showed that exposure to these compounds resulted in a rapid inhibition and, after removal of chemical agents, rapid recovery of GJIC occurs within a period of minutes that is not sufficient for the expression of adverse effects at the transcriptional level to occur. Connexins are integral membrane proteins with 4 transmembrane domains. The C terminal of connexin has a protein kinase motif, which suggests possible regulation by phosphorylation mechanisms (Kimura et al., 2000; Speisky et al., 1995
). However, previous results have indicated that no alteration in the phosphorylation of connexins is caused by PFFAs (Upham et al., 1998
) so that such a phosphorylation mechanism seems not to apply in this case (Hii et al., 1995
). Furthermore, induced changes in the phosphorylation of connexins do not always correlate with GJIC inhibition (Hossain et al., 1999
; Upham et al., 1997
). There are also several other examples where various compounds known to inhibit GJIC do not alter the phosphorylation status of the connexins and their underlying mechanism of action remains unknown (Sai et al., 2000
; Suzuki et al., 2000
; Upham et al., 2000
).
The results from the current study support a structure-activity relationship for the inhibitory effects of perfluorinated sulfonic acids on GJIC. Previous studies have shown that PFFAs, such as PFOA and PFDA, can inhibit GJIC in a dose-dependent manner. The inhibitory potency of PFFAs depends on the length of the carbon chain, PFFAs with carbon chain lengths less than 5 or more than 16 did not inhibit GJIC (Upham et al., 1998). In contrast, PFFAs with carbon chain lengths of 7, 8, 9, or 10 completely inhibit GJIC at concentrations of 50 µM (25 mg/l; Upham et al., 1998
). Our data are consistent with these previously published results. PFOS, which has an 8-carbon chain effectively inhibits GJIC, with an EC50 value of 36 µM (18 mg/l). PFOSA, the amide derivative of PFOS, inhibited GJIC with a similar potency to PFOS. However, it should be noted that the optimum chain length for the carboxyl fatty acids is 10 carbons while that for the sulfonic acids is 8 carbons. PFHS and PFBS, 6-carbon and 4-carbon chains respectively, but with the same functional group, do not inhibit GJIC. This indicates that the critical feature that determines GJIC inhibition for the PFFAs is the length of the carbon chain, not the nature of the functional group. This result suggests that the mode of action is based on a specific binding site for the ligands on the proteins of the gap junction since only ligands of a certain structure and size can elicit the observed effects.
It is significant that the structure-activity relationship for GJIC inhibition by endogenous fatty acids is different from that for the fluorinated analogues. As well as being less potent than the PFFAs, the optimum chain length for inhibition of GJIC by native fatty acids is 1618 carbons compared to the 89 optimum carbon chain length for PFFAs (Boger et al., 1998). In addition, the native fatty acids require a terminal carbonyl group capable of accepting a hydrogen bond so that the underivatized free fatty acids are essentially inactive (Boger et al., 1998
). This compares to the inhibition caused by the PFFAs, which is relatively insensitive to the nature of the functional group. Optimum activity of fatty acids also requires a delta-9 double bond and a hydrophobic methyl terminal group (Boger et al., 1998
). Together these observations indicate that the site of action of PFFA and native fatty acids for GJIC inhibition are different and that PFFAs are not simply acting as fatty acid analogues. A similar situation is observed with the binding of PFFAs to serum albumin where it appears PFFAs are bound to the protein at sites other than the fatty acid binding sites (Jones, unpublished results).
To date, most studies of GJIC inhibition have been conducted using the well-developed rat liver cell model. In this study dolphin kidney cells CDK were also used to test species specificity. Since inhibition of GJIC was observed in both cell lines the inhibitory effect of PFFAs on GJIC is neither species- nor tissue-specific. However, since the dolphin cells were from a different species and a different organ than the rat cells it is not possible to make direct comparisons. Thus dolphin kidney cells CDK could be used as an effective model for effects of perfluorinated compounds on GJIC in marine mammal species.
To understand more completely the environmental relevance and effects of PFOS, it is necessary to evaluate the same endpoints using in vivo exposure systems. Since it is not possible to conduct in vivo exposure on bottlenose dolphin, we conducted PFOS exposure in Sprague-Dawley rats. Although effects on GJIC in the parenchymal tissue of liver are primarily expressed through gap junctions, which contain Cx32 and 26, most tumor promoting compounds (e.g., phthalalte esters, 12-O-tetradecanoylphorbol-13-acetate, butylated hydroxytoluene, DDT, lindane, Aroclor 1254, clofibrate, trichloroethylene) that inhibit GJIC through Cx43 gap junctions are also known to inhibit GJIC in hepatocytes isolated from mouse and rat liver (Guan and Ruch, 1996; Jansen et al., 1996
; Klaunig et al., 1988
; Leibold et al., 1994
; Ruch et al., 1987
). Consistent with these in vitro studies, PFOS significantly inhibited GJIC in the livers of both treatment groups relative to the control, indicating that PFOS not only inhibits GJIC in Cx43 gap junctions but also in Cx32/26 gap junctions. This suggests the potential for PFOS to affect GJIC in multiple organisms. Oval cells, which have Cx43 gap junctions, are a major target for tumor promoting chemicals (Ruch and Trosko, 1999
), so it is unfortunate that there is no in vivo technique to measure GJIC in these cells that exist as small populations in the periportal regions of the liver. However, it is not unreasonable to suspect that these cells would also be affected in vivo since PFOS inhibits GJIC in these cells under in vitro conditions. The in vivo results also suggest that PFOS is a robust inhibitor of GJIC. The final mean accumulated doses of PFOS in rat liver samples after 3-day or 21-day exposure were 125.6 µg/g and 725.5 µg/g, respectively. However, no significant difference was observed in GJIC between short-term and long-term exposure. This could be explained by the fact that even after short-term exposure the accumulated dose was sufficient to cause maximum GJIC inhibition. Therefore extended exposure cannot cause further inhibition of GJIC. Even though the toxicokinetics of PFOS accumulation were expected to be different between male and female rats, the final dose of PFOS detected in samples of both male and female rat liver were similar. Furthermore, there was no significant difference in the measured inhibitory effects of PFOS on GJIC between male and female rats. This indicates that the effect of PFOS on cell-cell communication is not gender-related. Overall, these in vivo results suggest that PFOS poses a risk to the health of mammalian systems by interrupting GJIC, which is crucial in the maintenance of homeostasis within a tissue. Whether PFOS would pose a risk to human health cannot be determined, particularly, since many peroxisome proliferating compounds affect only rodents and not humans (Cattley et al., 1998
).
Together the results of this study demonstrate that, of the compounds tested, PFOS is the most potent inhibitor of GJIC activity and that its potency is equivalent to PFDA, the most potent GJIC-inhibitor of the carboxylic-PFFAs. Inhibition of GJIC was observed both in vitro and in vivo, demonstrating the relevance of GJIC inhibition to organisms exposed in vivo.
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ACKNOWLEDGMENTS |
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NOTES |
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