Exposure to common food additive carrageenan leads to reduced sulfatase activity and increase in sulfated glycosaminoglycans in human epithelial cells

2.2. Measurement of activity of sulfatases

Activity of several sulfatases was measured in NCM460 and MCF-7 cell homogenates at 80–90% confluence after 4–6 days in culture, as indicated in Tables 1 and ​and22 and in Results 3.1 and 3.2. Cells were exposed to λ-, κ-, or ι-carrageenan (1 ug/ml) (Fig. 1), and activity was compared to unexposed cells grown simultaneously under the same conditions. Methods used for assay of N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB), galactose-6-sulfatase (GALNS), steroid sulfatase (STS), iduronate-2-sulfatase (IDS), and arylsulfatase A (ARSA) were published previously [16]. Cell homogenates were prepared and assays were performed using at least triplicate biological samples with technical replicates of each measurement.

ARSB activity assay was performed by a fluorometric assay with 20 µl of cell homogenate, 80 µl of assay buffer (0.05 M Na-acetate buffer, pH 5.6), and 100 µl of substrate (5 mM 4-MUS in assay buffer) in wells of a microplate. The microplate was incubated for 30 min at 37 °C. The reaction was stopped by adding 150 µl of stop buffer (Glycine-Carbonate buffer, pH 10.7), and fluorescence was measured at 360 nm (excitation) and 465 nm (emission) (FLUOstar, BMG Labtech, Inc., Gary, NC). ARSB activity units are nmol/mg protein/hour, and were derived from a standard curve prepared with known quantities of 4-methylumbilleferyl at pH 5.6.

Substrates for determinations of activity of galactose-6-sulfatase (GALNS) and iduronate-2-sulfatase (IDS) were obtained (Moscerdam Substrates, Rotterdam, The Netherlands), and the assays were performed in accord with previously published protocols [18,19]. GALNS assay was performed with 5 µl cell homogenate made in ddH₂O by sonication with metal tip combined with 5 µl 0.2% heat-inactivated BSA (or 10 µl of 0.2% heat-inactivated BSA for blank) and 20 µl of substrate [10 mM 4-methylumbilliferyl-β-d-galactoside-6-sulfateNH₄ (MU-βGal-6S)] in substrate buffer [0.1 M sodium acetate/0.1 M acetic acid at pH 4.3 with 0.1 M NaCl, 5 mM Pb-acetate (1.9 mg/ml) and 0.02% Na-azide] in wells of a microtiter plate. The plate was sealed, and incubated for 17 h at 37 °C. Next, 5 µl 0.9 M Na-Phosphate buffer at pH 4.3 with 0.02% Na-azide was added, as well as 10 µl of 10 U β-d-Galactoside galactohydrolase (Sigma)/ml 0.2% heat-inactivated BSA. Reactants were incubated for 2 h at 37 °C, and then 200 µl of stop buffer [0.5 M NaHCO₃/0.5 M Na₂CO₃ at pH 10.7 with 0.025% Triton-X-100] was added. Fluorescence readings were taken at 360 nm and 465 nm. GALNS activity is expressed as nmol/mg protein/hour.

For IDS activity, 10 µl of cell homogenate made in water by sonification with a metal tip [or 10 µl 0.2% heat-inactivated bovine serum albumin (BSA) for the blank] was combined with 20 µl of substrate in substrate buffer (1.25 mM 4-MUS in 0.1 M Na-acetate/0.1 M acetic acid at pH 5.0 with 10 mM Pb-acetate). Cell homogenate, substrate, and substrate buffer were incubated 37 °C × 4 h. After 4 h, 40 µl of 0.4 M Na-Phosphate/0.2 M citrate buffer at pH 4.5 and 0.02% Na-azide were added, as well as 10 µl of LEBT (lysosomal enzymes purified from bovine testis), followed by incubation for 24 h at 37 °C. Next, 200 µl of stop buffer consisting of 0.5 M NaHCO₃/Na₂CO₃ at pH 10.7 with 0.025% Triton-X-100 was added, and readings were taken at 360 nm and 465 nm. IDS activity is expressed as nmol/mg protein/hour.

ARSA activity was determined by a spectrophotometric technique using p-nitrocatechol sulfate (NCS) as substrate, and based on the retention of ARSA activity at low temperature, in contrast to ARSB activity [20,21]. Pre-chilled reagents were used, and cells were homogenized in ddH₂O by sonication. Cell homogenates were centrifuged for 10 min at 5000 g at 4 °C, and the supernatant was used for the enzyme activity assay. Cell extracts were incubated in a total volume of 200 µl 0.5 M Na-acetate buffer at pH 5.0 with 200 µl of 10 mM p-NCS in an ice-bath. After 17 h, the reaction was stopped by adding 600 µl of 1 M NaOH, and the reaction mixture was centrifuged for 5 min to remove precipitated material. The optical density of the clear supernatant was measured at 516 nm in a Beckman spectrophotometer. ARSA activity is reported as nmol/mg protein/hour.

STS activity assay was performed using 20 µl of cell homogenate, 80 µl of assay buffer, and 100 µl of substrate [16,22]. The substrate was 0.5 mM 4-methylumbilleferyl-sulfate (4-MUS) in substrate buffer consisting of 0.5 M Tris-C Buffer at pH 7.5. The reaction mixture was incubated for 4 h at 37 °C, then 100 µl of stop buffer (1 M Tris-Cl buffer at pH 10.4) was added. Fluorescence was measured at 360 nm and 465 nm (FLUOstar, BMG Labtech Inc., Cary, NC). STS activity units are nmol/mg protein/hour, and were calculated by a standard curve prepared with known quantities of 4-methylumbilleferyl at pH 7.5.

Measurement of heparan sulfamidase activity was performed following an established assay procedure [23], using the substrate methylumbilleferyl-α-d-N-sulphoglucosaminide.Na (MU-αGlcNS), the substrate buffer consisting of Na-barbital in Na-acetate prepared with 0.7% NaCl at pH 6.5 with 0.02% (w/v) Na-azide, and the protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl-fluoride. HCL (Roche, Indianapolis, ID). α-Glucosidase from Bacillus stearothermophilus (Sigma Chemical Company, St. Louis, MO) 10 U/ml BSA-0.2%, double concentrated McIlvains phosphate/citrate (Pi/Ci) buffer, and bicarbonate stop buffer at pH 10.7 with 0.025% Triton-X-100 were required. Cell homogenates (30 µg protein) were prepared in water by sonification with a metal tip. Cell preparations were incubated with 10 µl heat denatured BSA-0.2%, 10 µl MU-αGlcNS and 10 µl of the protease inhibitor for 17 h at 47 °C. Then the reaction was stopped with 6 µl Pi/Ci buffer and mixed with 10 µl α-glucosidase and incubated for 24 h at 37 °C. The reaction was terminated by addition of 200 µl of the stop buffer, and the fluorescence of the MU read. Activity was expressed as nmol/mg protein/hour.

SULF-1 and SULF-2 are endosulfatases that act extracellularly to remove sulfate groups from heparin and heparin sulfate. Activity was measured in the spent media of the NCM460 cells following exposure to λ-carrageenan or unexposed control following an established procedure [24]. Concentrated spent media were mixed with 10 mM 4-MUS, 10 mM lead acetate, and 50 mM HEPES at pH 8.0 yielding a total volume of 30 µl. The mixture was incubated for 2 h at 37 °C. 100 µl of 0.5 M Na₂CO₃/NaHCO₃ was used to stop the reaction, and fluorescence was measured, the OD was correlated with known concentrations of 4-MU at pH 8.0, and the activity expressed as nmol/mg protein/h.

2.3. Cell-based ELISA and cDNA microarray of sulfatases

Cell-based ELISA is designed as a microplate-based Western blot (Active Motif, Carlsbad, CA), and was used to detect the protein content of ARSB, ARSA, GALNS, and STS in NCM460 and T84 cells after exposure to λ-carrageenan for 48 h [10]. Activity at 48 h was determined by the procedures described above and were within the standard deviation of the activity at 4 days that is reported in Table 1 for these enzymes. Antibodies used for detection were ARSB rabbit polyclonal antibody (1:250; Open Biosystems, Huntsville, AL), GALNS rabbit polyclonal antibody (1:250; Open Biosystems, Huntsville, AL), STS mouse monoclonal (2 µg/ml, Abcam, Cambridge, MA) and ARSA mouse monoclonal (4 µg/ml; R&D, St. Paul, MN). Rabbit or mouse IgG was used for control experiments. For the cell-based ELISAs, NCM460 or T84 cells were seeded at densities of 2500, 5000, and 10,000 cells per well in a 96-well plate with n = 5 for each of the determinations. After 48 h, the media were aspirated, the cells were washed with PBS, and then fixed ×20 min at room temperature with 100 µl of formaldehyde 4% in PBS. The formaldehyde was removed and cells were washed with wash buffer (0.1% Triton-X-100 in PBS) 200 µl three times, wash buffer was aspirated, and cells were incubated with quenching buffer (1% H₂O₂ in wash buffer) 100 µl for 20 min at room temperature. Then quenching buffer was removed and cells were washed three times with wash buffer 200 µl. Wash buffer was aspirated and cells were incubated with blocking buffer (PBS with 5% non-fat milk/FBS) 100 µl to each well for 1 h at room temperature. Blocking buffer was aspirated, and primary antibody to either ARSB, ARSA, GALNS, or STS was added (diluted in PBS with 0.1% heat-inactivated BSA), and the plate the sealed and incubated for 2 h at room temperature. Primary antibody was aspirated, and cells were washed with 200 µl of wash buffer three times, and the secondary antibody (100 µl) was added to each well, the plate was sealed and incubated for 1 h at room temperature. The secondary antibody was aspirated and cells were washed with wash buffer (200 µl) three times, then 100 µl of developing solution was added to each well, and incubated at room temperature ×15 min and protected from light. Stop solution (2N sulfuric acid; 100 µl) was added to each well, then absorbance was read at 450 nm. Cells were washed with 200 µl of wash buffer ×3, excess liquid was removed from the wells, and the plates were air-dried at room temperature for 5 min. Crystal violet solution 100 µl was added to each well, and the plates were incubated for 30 min at room temperature, washed with wash buffer three times, and 100 µl SDS solution was added to each well, and the plate was incubated on a shaker for 1 h at room temperature and final absorbance was read at 595 nm. The linearity of the determinations was confirmed by comparisons of the intensity at three different cell concentrations.

cDNA microarrays were performed on NCM460 cells after exposure to λ-carrageenan (1 µg/ml) for 4 days using the Affymetrix Human Gene Chip U133 plus 2.0 system (Santa Clara, CA) [25]. Total RNA was extracted using the RNeasy minikit (Qiagen) from replicate biological samples of carrageenan-treated and control samples. Each chip contained 11 probeset pairs (22 probes total) for each gene with 54,613 transcripts present on the chip. Pair-wise comparisons of control vs. carrageenan-treated mean fluorescent intensity values were performed, and a total of 790 transcripts had uncorrected P-values less than 0.001.

2.4. Materials and reagents for disaccharide analysis

Actinase E was from Kaken Biochemicals (Tokyo, Japan). Chondroitin lyases ABC and ACII and heparin lyases 1, 2 and 3 were from Seikagaku Corporation (Tokyo, Japan). Urea, CHAPS were from Sigma Chemical Company (St. Louis, MO, USA). Vivapure MINI Q H spin columns were from Viva science (Edgewood, NJ, USA). Microcon Centrifugal Filter Units YM-3 was from Millipore Company (Bedford, MA, USA). Unsaturated disaccharide standards of CS/DS (ΔDi-0S: ΔUA-GalNAc, ΔDi-4S: ΔUA-GalNAc4S, ΔDi-6S: ΔUA-GalNAc6S, ΔDi-2S: ΔUA2S-GalNAc, ΔDi-diSB: ΔUA2S-GalNAc4S, ΔDi-diSD: ΔUA2S-GalNAc6S, ΔDi-diSE: ΔUA-GalNAc4S6S, ΔDi-triS: ΔUA2S-GalNAc4S6S) and unsaturated disaccharides standards of HP/HS (ΔDi-0S: ΔUA-GlcNAc, ΔDi-NS: ΔUA-GlcNS, ΔDi-6S: ΔUA-GlcNAc6S, ΔDi-2S: ΔUA2S-GlcNAc, ΔDi-2SNS: ΔUA2S-GlcNS, ΔDi-NS6S: ΔUA-GlcNS6S, ΔDi-2S6S: ΔUA2S-GlcNAc6S, ΔDi-triS: ΔUA2S-GlcNS6S) were obtained from Seikagaku Corporation (Japan). All other chemicals were of regent grade.

The molecular weights (MW) of the CS disaccharides are: Di-0S (UA-GalNAc) 379; Di-4S(UA-GalNAc4S) 459; Di-6S(UAGalNAc6S) 459; Di-2S(UA2S-GalNAc) 459; Di-diSB(UA2S-GalNAc4S) 539; Di-diSD(UA2S-GalNAc6S) 539; Di-diSE(UA-GalNAc4S6S) 539; and Di-triS(UA2SGalNAc4S6S) 637. The MW of the HS disaccharides are: Di-0S(UA-GlcNAc) 379; Di-NS(UA-GlcNS) 417; Di-6S(UA-GlcNAc6S) 458; Di-2S(UA2S-GlcNAc) 458; Di-2SNS(UA2S-GlcNS) 497; Di-NS6S(UA-GlcNS6S) 497; Di-2S6S(UA2S-GlcNAc6S) 537; and Di-triS(UA2S-GlcNS6S) 576. The molecular weights of the carrageenan disaccharides are different than those of the GAGs disaccharides. The carrageenan-derived disaccharides consist of 3,6-anhydro-galactose (A-Unit) and galactose (G-Unit) or two galactose (G-unit). The MW of the carrageenan-derived disaccharides are: kappa-carra-disaccharide (A-G4S) 404; iota-carra-disaccharide (A2S-G4S) 484; and lambda-carra-disaccharide (G4S-G2S6S) 582.

2.5. Isolation and purification of GAGs from cells

The cell samples (in 1 ml water) were individually subjected to proteolysis at 55 °C with 10% of actinase E (20 mg/ml) for 20 h. After proteolysis, dry urea and dry CHAPS were added to each sample (2 wt% in CHAPS and 8 M in urea). Particulates were removed from the resulting solutions by passing each through a syringe filter containing a 0.22 µm membrane. A Vivapure MINI Q H spin column was prepared by equilibrating with 200 µL of 8 M urea containing 2% CHAPS (pH 8.3). The clarified filtered samples were loaded onto and run through the Vivapure MINI Q H spin columns under centrifugal force (2000 × g). The columns were first washed with 200 µL of 8 M urea containing 2% CHAPS at pH 8.3. The columns were then washed five-times with 200 µL of 200 mM NaCl. GAGs were released from the spin column by washing three times with 50 µL of 16% NaCl. GAGs were desalted with a Microcon Centrifugal Filter Units YM-3 (3000 MWCO) spin column. The GAGs were freeze-dried for future use.

2.6. Quantification of glycosaminoglycans

The isolated glycosaminoglycans (GAGs) were subjected to carbazole assay to quantify the amount of GAGs in each sample using heparan sulfate as standard. A standard curve of the heparan sulfate gave the equation y = 0.1065x+0.0498, R² = 0.9986. The amount of GAGs was determined in samples of 10⁷ cells treated with carrageenan for 4 days. The carbazole assay measures the hexuronic containing GAGs, including unsulfated GAGs (e.g. hyaluronan) and disaccharides, but not keratan sulfate or its digests.

The total sulfated GAGs, including chondroitin-4-sulfate (C4S), chondroitin-6-sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, and heparin, but not including disaccharides, were measured in cell lysates by sulfated GAG assay (Blyscan™, Biocolor Ltd, Newtownabbey, Northern Ireland). The cationic dye 1,9-dimethylmethylene blue reacts with the sulfated GAG, producing an insoluble dye-GAG complex, and the sGAG content is determined by the amount of dye recovered from the test sample following exposure to Blyscan Dissociation Reagent. The ratio of the GAG dye-binding capability to the C4S sulfation level is reported as 1.0 for C4S from bovine trachea [26]. Absorbance maximum of 1,9-dimethylmethylene blue is 656 nm. Concentration by Blyscan assay is expressed as µg/mg protein of cell lysate. C4S was determined by this assay following immunoprecipitation of cell lysates from treated and control cells using the C4S (4D1) antibody (Abnova) to detect native C4S, as previously described [27].

2.7. Disaccharide composition analysis using LC–MS

GAG samples (5 µg) were incubated with the chondroitinase ABC (10 m-units) and chondroitinase ACII (5 m-units) at 37 °C for 10 h [28,29]. The enzymatic products were recovered by centrifugal filtration (YM-30, 30,000 MWCO, Millipore, Bedford, MA). CS/DS disaccharides passed through the filter, were freeze-dried and ready for LC–MS analysis. Next, the heparinases I, II and III (5 mU each) were add into the remainder and incubated at 37 °C for 10 h. The products were again recovered by centrifugal filtration and the heparin/HS disaccharides were similarly collected and freeze-dried and ready for LC–MS analysis.

MS analyses were performed on an Agilent 1100 LC/MSD instrument (Agilent Technologies, Inc. Wilmington, DE) equipped with an ion trap, binary pump followed by microflow, and a UV detector. For CS/DS disaccharide analysis, the column used was a 1.7 µm Acquity UPLC BEH C18 column (2.1 × 150 mm, Waters, Milford, MA, USA). Solutions A and B for UPLC were 0 and 75% acetonitrile, respectively, containing the same concentration of 15 mM hexylamine as an ion-pairing reagent and 100 mM hexa-fluoroisopropanol as an organic modifier. The column temperature was maintained at 45 °C. For disaccharide analysis, solution A for 10 min was followed by a linear gradient from 10 to 40 min of 0–50% solution B at the flow rate of 100 µL/min. The electrospray interface was set in positive ionization mode with the skimmer potential 40.0 V, capillary exit 40.0 V, and a source of temperature of 350 °C to obtain maximum abundance of the ions in a full-scan spectra (350–2000 Da, 10 full-scans/s). Nitrogen was used as a drying gas (8 L/min) and a nebulizing gas (40 psi).

For HS/HP disaccharide analysis, eluent A was water/acetonitrile (85:15) v/v, and eluent B was water/acetonitrile (35:65) v/v. Both eluents contained 12 mM tributylamine (TrBA) and 38 mM NH₄OAc with pH adjusted to 6.5 with HOAc. The column temperature was maintained at 45 °C. Solution A for 10 min, followed by a linear gradient from 10 to 40 min of 0–50% solution B at the flow rate of 100 µL/min, was used for disaccharide analysis. The electrospray interface was set in negative ionization mode with the skimmer potential −40.0 V, capillary exit −40.0 V, and a source of temperature of 350 °C to obtain maximum abundance of the ions in a full-scan spectra (350–1500 Da, 10 full-scans/s). Nitrogen was used as a drying gas (8 L/min) and a nebulizing gas (40 psi).

3.2. Sulfatase activity in human mammary cells following carrageenan

Consistent with the observed effects of carrageenan exposure on sulfatase activity in the human colonic epithelial cells, sulfatase activity declined significantly in several human mammary cell lines, including MCF-7, MCF-10A, T47D, HCC1937, as well as primary myoepithelial and epithelial cells (Table 2) (p < 0.001) following exposure to λ-carrageenan (1 µg/ml × 6 days). ARSB, ARSA, STS, GALNS, and IDS all declined following exposure to λ-carrageenan (p < 0.001). As reported previously, baseline activity of ARSB, ARSA, STS, and GALNS was higher in the normal, primary epithelial and myoepithelial cells than in the malignant, metastatic cell lines (MCF-7 and T47D), and sulfatase activity in the MCF-10A cell line was reduced for ARSB, ARSA, and GALNS, but STS and IDS activity was similar to the activity in the primary mammary cells [16]. Carrageenan exposure produced significant reductions in activity of each sulfatase in the different cell types (p < 0.001 or p < 0.01). As in the colonic cells, exposure to λ-carrageenan produced greater declines than κ- or ι-carrageenan in ARSB activity in the MCF-7 and MCF-10A cells, although all declines were highly significant (p < 0.001).

3.3. GAG content following carrageenan in NCM460 cells

The GAGs detected by carbazole assay are the uronic acid containing disaccharides and glycosaminoglycans, including hyaluronan (HA), a nonsulfated GAG, but excluding keratan sulfate, a sulfated GAG not containing an uronic acid residue. NCM460 cells exposed to kappa, iota or lambda-carrageenan for 4 days and unexposed cell control were isolated by a three-step procedure involving protease digestion, strong anion-exchange chromatography on a spin column, followed by salt release. Under the conditions used in the strong anion-exchange spin column step, unsulfated HA is poorly recovered. The GAG samples were subjected to carbazole assay to quantify the amount of GAG in each sample of 10⁷ cells (~1 mg protein/10⁶ cells). The GAG content from cells exposed to carrageenans was markedly higher than GAGs from the unexposed cell control (Table 3A) by carbazole assay.

The total sulfated GAG and the C4S content, determined by 1,9-dimethylmethylene blue assay, also demonstrated significant increases following carrageenan exposure (Table 3B). The differences between the two assays reflect differences in specific GAGs and degradation products, and suggest that the effects of ι-carrageenan exposure on the accumulation of unsulfated GAGs are greater than the impact of either λ- or κ-carrageenan.

The authors acknowledge the contributions of Dr. Anuj Jain to performance of the cell-based ELISA experiments.