Photoautotrophically grown Chlorella vulgaris show genotoxic

Jul 4, 2019 - C. vulgaris significantly induced DNA damage in both cell lines at a concentration of 200 µg dry matter/mL (comet tail intensity CTI: 2...
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Photoautotrophically Grown Chlorella vulgaris Shows Genotoxic Potential but No Apoptotic Effect in Epithelial Cells Andrea Gille,†,* Andreas Trautmann,‡ Manuel Rodriguez Gomez,† Stephan C. Bischoff,§ Clemens Posten,‡ and Karlis Briviba†

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†

Department of Physiology and Biochemistry of Nutrition, Max Rubner-Institut, Federal Research Institute of Nutrition and Food, 76131 Karlsruhe, Germany ‡ Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life Sciences III Bioprocess Engineering, 76131 Karlsruhe, Germany § University of Hohenheim, Institute of Nutritional Medicine, 70599 Stuttgart, Germany ABSTRACT: This study investigated the effect of Chlorella vulgaris (C. vulgaris) on genotoxicity, cytotoxicity, and apoptosis in Caco-2 and HT-29 cells. C. vulgaris significantly induced DNA damage in both cell lines at a concentration of 200 μg dry matter/mL (comet tail intensity CTI: 24.6 ± 4.7% for Caco-2, 16.6 ± 0.9% for HT-29). The application of processing (sonication, ball-milling) did not affect the genotoxicity negatively and lowered the lipid peroxidation in C. vulgaris preparations. C. vulgaris-induced intracellular formation of reactive oxygen species in human cell lines and might be responsible for the genotoxic effect. A solid fraction mainly triggered the observed DNA damage (CTI: 41.5 ± 1.9%), whereas a hydrophilic (CTI: 7.9 ± 1.7%) and lipophilic (CTI: 10.2 ± 2.1%) fraction revealed a significantly lower tail intensity. C. vulgaris significantly induced DNA damage in both cell lines possibly through intracellular formation of reactive oxygen species; however, it was repaired after a 2 h recovery time or was even avoided at lower concentrations. In addition, none of the preparations indicated an adverse effect on cell proliferation or revealed apoptotic activity. KEYWORDS: Chlorella, functional food, DNA damage, Caco-2 cells, HT-29 cells, processing toxicological parameters have already been observed.10,11 In contrast, numerous in vivo studies reported no adverse, toxic, or mutagenic effects related to the consumption of preparations containing different Chlorella species.10,12−15 Hence, data on safety evaluation are inconsistent and microalgae-based research should focus more intensively on potential adverse or toxic effects in the future. Several in vitro methods can be applied in terms of measuring toxicity related cell physiological end points. The depicted work focused on the genotoxicity of C. vulgaris preparations as assessed by the comet assay. This method determines the DNA damaging response in single cells.16,17 Fragmentation of DNA can also be a sign of apoptosis.18 Though primary human intestinal cells would represent a perfect model, we used the human colon epithelial cell lines Caco-2 and HT-29, since they have been frequently used in toxicity testing of food products.19−21 Especially HT-29 cells were preferably applied to test inductions of apoptosis and necrosis. Different methods have been established to assess the apoptotic effect such as the measurement of the activation of capase-322,23 or the external translocation of phosphatidylserine by binding of fluorophore-tagged Annexin V.24,25 The aim of the present study was to evaluate the cytotoxic, genotoxic, and pro-apoptotic/necrotic potential of different C.

1. INTRODUCTION The green microalgae Chlorella has been traditionally used for human nutrition and medicinal applications in Asian and African countries. Numerous health promoting effects have been discussed due to a high content of bioactive compounds such as micronutrients, minerals, vitamins, and phytochemicals (e.g., carotenoids).1,2 In recent decades, the nutritional value and application of microalgae in food have been extensively investigated and microalgae have been commercialized widely as food or dietary supplements in Japan, China,1,3 or the United States (U.S.).1 The supplementation with Chlorella vulgaris (C. vulgaris) in food is not subjected to the Novel Food Regulation (European Union, No. 258/97), since it was already consumed to a significant degree before 1997. Hence, Chlorella-derived products are already on the market, even though its toxic potential has not been investigated completely and its use for human nutrition is mainly based on traditional consumption. Moreover, the use of subfractions and processing methods for food application needs to be assessed separately.1 The influence of culture conditions on biomass composition was already shown in previous studies. Especially the macromolecular biomass composition, e.g., proteins, lipids, and carbohydrates, varies with the availability of nutrients and light.4−7 In general, a high nutritional value for Chlorella was proposed and no hints for the presence of toxins or allergens were declared.1 Nevertheless, safety hazards regarding the consumption of Chlorella are assessed rarely or not in accordance with the required guidelines. However, adverse health effects8,9 as well as information regarding relevant © XXXX American Chemical Society

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June 3, 2019 July 2, 2019 July 4, 2019 July 4, 2019 DOI: 10.1021/acs.jafc.9b03457 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry vulgaris preparations in Caco-2 and HT-29 cells. Moreover, we investigated the processing effects by sonication and ballmilling on these parameters.

2. MATERIALS AND METHODS 2.1. Chemicals. Chemicals for microalgae cultivation (ammonium heptamolybdate tetrahydrate, ammonium chloride, boric acid, calcium chloride dihydrate, cobalt(II) chloride hexahydrate, copper(II) sulfate pentahydrate, dipotassium phosphate, EDTA, iron(II) sulfate heptahydrate, magnesium sulfate heptahydrate, manganese(II) chloride tetrahydrate, TRIS, potassium dihydrogen phosphate, zinc sulfate heptahydrate) were obtained from Carl Roth (Karlsruhe, Germany) or Merck (Darmstadt, Germany). Eagle’s minimal essential medium (EMEM), Dulbecco’s minimal essential medium (DMEM), and phosphate-buffered saline (PBS, pH 7.4) were supplied by Lonza (Verviers, Belgium); nonessential amino acids (NEA) and trypsinEDTA (0,25%) by Fischer Scientific (Schwerte, Germany), penicillin/ streptomycin (P/S) and glutamine by Biozym (Hessisch Oldendorf, Germany); and fetal calf serum (FCS) by Biowest (Nuaillé, France). EDTA disodium salt (Na2EDTA), laurosyl sarcosine sodium salt, dichloro-dihydro-fluorescein diacetate, malondialdehyde, xylenol orange, trypan blue, staurosporin, brefeldin A, and camptothecin were purchased from Sigma-Aldrich (Taufkirchen, Germany). Normal melting agarose was obtained from FMS (Rockland, U.S.A.), and low melting agarose was obtained from Cambrex (Rockland, U.S.A.). Hydrogen peroxide (H2O2) and sodium hydroxide (NaOH) were acquired from Merck (Darmstadt, Germany); magnesium sulfate heptahydrate and sodium ammonium hydrogen phosphate tetrathydrate as well as Kobe I agar-agar, D-glucose, sodium chloride, monosodium phosphate, and sodium hydrogen phosphate were obtained from Carl Roth (Karlsruhe, Germany). 2.2. Microalgae Cultivation and Biomass Harvesting. C. vulgaris (wildtype 211-12), obtained from the culture collection of algae (SAG, University of Goettingen, Germany), was cultivated and further processed as described previously.26 In brief, liquid cultures of C. vulgaris were cultivated in shaking flasks (100 rpm) containing TAP medium (acetate-free; pH 7.5)27 at 25 °C under continuous illumination (160 μmol m−2s−1). The biomass was harvested by centrifugation of the liquid cultures, the supernatant was discarded, and the remaining biomass was stored at −20 °C. 2.3. C. vulgaris Preparations. Aliquots containing 20 mg of C. vulgaris dry matter (dm)/10 mL HBSS were prepared and either stored immediately at −20 °C until use (unprocessed) or sonicated. As described previously,26 sonication was performed using the following conditions: 15 min, 5 cycles/min, frequency of 20 kHz, ultrasonic intensity of 9.8 W cm−2 in a SONOPULS HD 2070 homogenizer from Bandelin (Berlin, Germany). The samples were subsequently stored at −20 °C after sonication. Prior to ball-milling, C. vulgaris biomass was lyophilized while being protected from light by using a Christ Alpha 1−2 LD freeze drier (Osterode a. Harz, Germany). Then, the lyophilized samples were resuspended in deionized water and treated by using a ball mill MM 300 from Retsch (Haan, Germany) at a shaking frequency of 30 Hz for 10 min after addition of 3 stainless steel beads (3 mm diameter). The samples were subsequently stored at −20 °C. Subcellular Fractionation by Centrifugation. The fractionation by centrifugation (Figure 1) was performed as described by previous authors with slight modifications.28,29 First, the suspension consisting of ball-milled C. vulgaris (20 mg dm/10 mL) was centrifuged at 1500 × g for 5 min at 4 °C. The pellet (P1) was stored at −20 °C until utilized for cell treatment, whereas the supernatant (S1) was centrifuged a second time at 10000 × g for 30 min at 4 °C. Thereby, a pellet (P2) consisting of cell organelles and a supernatant (S2) representing the cytosol were obtained. P1 and P2 were resolved in 10 mL of HBSS for incubation of Caco-2 cells (Figure 1). Subcellular Fractionation by Folch Extraction. The extraction method according to Folch et al. was used to separate components due to their solubilities in different solvents.30 Therefore, 20 mg of ball-milled C. vulgaris biomass was solved in 5 mL of HBSS; the

Figure 1. Centrifugation method to separate distinct subcellular fractions of C. vulgaris. solution was mixed thoroughly, laced with methanol, and mixed again for 2 min. Then, 6 mL of chloroform was added, and the solution was mixed again thoroughly for 2 min. The mixture was centrifuged for 10 min at 4000 × g; the upper (methanol and HBSS; hydrophilic) phase, the lower (chloroform; lipophilic) phase, and the solid phase were transferred separately to new tubes followed by evaporation of the solvents. For Caco-2 cell treatment, the fractions were resolved in 10 mL of HBSS containing 0.05% tetrahydrofuran. 2.4. Caco-2 Cell Culture and Cell Treatment. Caco-2 and HT29 colon carcinoma cells were obtained from the German collection of Microorganisms and Cell Cultures (DMSZ, Braunschweig, Germany). Caco-2 cells were cultivated in EMEM (supplemented with 10% (v/v) FCS, 1% (v/v) glutamine, 1% (v/v) NEA, and 1% (v/ v) P/S), whereas HT-29 cells were grown in DMEM (supplemented with 10% (v/v) FCS, 1% (v/v) NEA and 1% (v/v) P/S) at 37 °C in humidified air with 5% (v/v) CO2. To perform the comet assay, WST1 test, and apoptosis/necrosis assays, the cells were cultured in 6well plates (Caco-2 cells: 0.02 * 106/well; HT-29 cells:0.1 * 106/ well), whereas ROS formation was assessed in 96-well plates (Caco-2 cells: 1 * 103/well; HT-29 cells:5 * 103/well). Both cell lines were grown for 5−6 d with growth medium being changed every other day. The assays were performed at a cell confluence of 70−85%. To achieve differentiation, Caco-2 cells were grown for at least 18 d in 6well plates (0.02 * 106/well). The growth medium was changed every other day, and the differentiated cells were used for experiments between day 18 and 21. Prior to cell treatment, the C. vulgaris suspensions were diluted to a concentration of 200 and 20 μg C. vulgaris dm/mL. To assess genotoxic effects, Caco-2 and HT-29 cells were incubated for 2 h with pure HBSS (containing CaCl2, diluent/negative control), the C. vulgaris preparations, or 100 μM H2O2 (positive control). Then, the incubation solutions were aspirated and the cells were washed twice with PBS and used to determine the induction of DNA damage. To assess the time-dependent repair of C. vulgaris-induced DNA damage and the effect on cell viability/proliferation (WST-1 test), the Caco-2 cells were incubated for 2 h with C. vulgaris (200 μg dm/mL) and for an additional 2 or 24 h with EMEM (complete medium). In several prestudies, the optimal incubation times were determined for both cell lines with several positive substances in order to observe an apoptotic effect (Annexin V-staining and caspase3 activation). Caco-2 as well as HT-29 cells were treated with the appropriate positive controls or C. vulgaris preparations in a concentration of 20 μg dm/mL for 24 h (HT-29 cells), 48 h (undifferentiated Caco-2 cells), or 4 h (differentiated Caco-2 cells). Afterward, the cells were harvested to measure apoptosis and necrosis. The cell viability was assessed throughout all experiments by trypan blue staining (blue staining of DNA in necrotic cells). Therefore, the cells were trypsinized with 1 mL trypsin/EDTA, stained with trypan blue, and counted in a hemocytometer. B

DOI: 10.1021/acs.jafc.9b03457 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry 2.5. Single Cell Microgel Electrophoresis Assay (Comet Assay). Damage of cellular DNA was determined with the single cell gel electrophoresis assay (comet assay) as described by Briviba et al.31 Here, Caco-2 or HT-29 cells (2 * 105 cells) were mixed with 85 μL of 0.7% low melting point agarose and added to a microscope slide covered with 0.5% normal melting agarose. The slides were placed in lysis buffer (100 mM Na2EDTA, 1% v/v Triton X 100, 2.5 mM NaCl, 1% v/v lauroyl sarcosine sodium salt, 10% v/v dimethyl sulfoxide, 10 mM Tris, pH 10) for 1 h, followed by placement in an alkaline electrophoresis buffer (1 mM Na2EDTA, 300 mM NaOH; pH 13) for 20 min to allow unwinding of DNA. Electrophoresis was performed at 25 V, 300 mA for 40 min. Finally, the DNA was stained with the fluorescence dye DAPI (4′,6-Diamidin-2-phenylindol; 0.5% (w/v)); 50 randomly selected cells were analyzed by fluorescent microscopy (DM 400 B, Leica Microsystems; Mannheim, Germany) and quantified by the imaging software of Perceptive Instruments (Halstead, U.K.). The extent of DNA damage is expressed as the percentage of DNA in the comet tail (tail intensity). 2.6. Cell Viability and Proliferation (WST-1 Assay). Cell viability and proliferation was assessed by the WST-1 (2-(4iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsodiumsalt) assay according to the manufacturer’s protocol (Roche Diagnostics GmbH, Mannheim, Germany). Following incubation with test solutions or EMEM, the cells were incubated with WST 1 solution for 1 h. The absorbance of the formed formazan was measured at 450 nm (reference wavelength 690 nm) using a Tecan microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany). 2.7. ROS Measurement (DCFH-DA Assay). The pro-oxidative activity of the C. vulgaris preparations was measured by using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) probe. Here, growth medium was discarded; the cell layer was washed once with 150 μL of PBS and then incubated for 30 min with 100 μL of a 10 μM DCFH-DA solution (in HBSS). Subsequently, the cells were washed again with 150 μL of PBS and treated for 2 h with HBSS (containing CaCl2, diluent control), with C. vulgaris preparations or the positive controls tert-butylhydroperoxide (TBHP) and H2O2 at a concentration of 100 μM. After incubation, the fluorescence was measured at 485 nm (excitation) and 530 nm (emission) using a Tecan microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany). To exclude interferences of the measurement by the C. vulgaris preparations, the fluorescence emission of each test preparation was determined using the same conditions. For blank measurements, cells were exposed to HBSS instead of the DCFH-DA probe and then incubated with the test preparations. 2.8. Determination of Lipid Peroxidation Products. Thiobarbituric Acid Reactive Substances (TBARS). TBARS were measured according to the method describe previously with slight modifications.32 To a 500 μL sample (C. vulgaris suspension), 10 μL of butylated hydroxytoluene solution (10 mM, dissolved in ethanol), 350 μL of trifluoroacetic acid (20%, v/v), and 500 μL of thiobarbituric acid (1.4%, w/v) were added, and the mixture was incubated at 95 °C for 45 min. Afterward, the mixture was centrifuged for 10 min (16000 × g) and the supernatant was analyzed via HPLC. The HPLC system was equipped with a pump, auto sampler, column oven, and fluorescence detector from VWR-Hitachi as well as a Luna C18 LC column (5 μM, 250 × 4.6 mm) from Phenomenex (Aschaffenburg, Germany). The mobile phase consisted of potassium phosphate (0.02 mM; pH 6.5) and methanol (65/35, v/v), and the flow rate was set to 0.75 mL/min. Standard solutions of malondialdehyde (MDA) in a range of 0.1−2 μM were used for calibration. The resulting peak was detected with a fluorescence detector (excitation and emission wavelengths, 515 and 550 nm, respectively). Ferrous Oxidation Xylenol Orange (FOX)-Assay. The FOX-assay was applied according to the method of Gay et al.33 with minor modifications. This method is not specific for H2O2; therefore, lipid as well as water-soluble peroxides can be detected when using the fractions obtained by Folch extraction. For this reason, both subfractions were applied as methanolic solutions to the test

(chloroform was evaporated and the residue resolved in an equivalent volume of methanol). Catalase (Cat) and tris(2-carboxyethyl)phosphine (TCEP) were added to examine the potential decompensation or reduction of existing peroxides by preincubating with 40 μL of a 200 U/mL Cat solution or 40 μL of a 80 mM TCEP solution. To a 40 μL sample, 40 μL of iron(II) sulfate (1.5 mM, in 25 mM sulfuric acid) and 280 μL of xylenol orange solution (150 μM, in 25 mM sulfuric acid) were added, and the mixture was incubated for 30 min at room temperature. After incubation, 100 μL of the mixture was transferred to one well of a 96-well plate and the absorbance was measured at 560 nm (Tecan microplate reader, Tecan Deutschland GmbH, Crailsheim, Germany). 2.9. Measurement of Apoptosis and Necrosis. In order to assess the induction of apoptotic signals in Caco-2 and HT-29 cells, the activation of intracellular caspase-3 as well as the translocation of phosphatidylserine to the outer leaflet was determined. Therefore, the FITC Active Caspase-3 Apoptosis Kit from BD Pharmingen (Heidelberg, Germany) and the PE Annexin V Kit from BioLegend (Koblenz, Germany) were used as described in the manufacturers’ protocol. Moreover, in combination with the PE Annexin V, necrosis was determined by the Zombie Violet Fixable Viability Kit from BioLegend (Koblenz, Germany). In brief, after treatment, cells of one well (HT-29 cells, differentiated Caco-2 cells) or 2 wells (undifferentiated Caco-2 cells) of a 6-well plate were trypsinized, washed once with PBS, and resuspended in 2 mL of ice-cold PBS. First, 100 μL of a Zombie Violet dye working solution (dilution: 1/ 1000 in DMSO) were added. After a light-protected incubation for 15 min at room temperature, the suspension was centrifuged (250 × g, room temperature, 5 min), and the pellet was washed twice with icecold PBS. For Annexin V-staining, the cell pellet was resuspended in 100 μL of Annexin V Binding Buffer, followed by the addition of 5 μL of PE Annexin V and an incubation for 5 min at room temperature in the dark. Then, the cells were washed twice with ice-cold PBS, the pellet was resuspended in 500 μL of BD Cytofix/Cytoperm solution, and the solution was incubated for 20 min on ice in the dark for permeabilization. The solution was centrifuged (250 × g, room temperature, 5 min), the cell pellet was washed twice with 500 μL of BD Perm/Wash buffer, and it was resuspended in 100 μL of BD Perm/Wash buffer. Next, 20 μL of FITC rabbit Anti-Active Caspase-3 was added, and the samples were incubated for 30 min at room temperature under light protection. The cells were then washed once with 1 mL of BD Perm/Wash buffer and resuspended in 500 μL BD Perm/Wash buffer; samples were stored on ice until measurement with a FACSVerse flow cytometer (Becton Dickinson, Heidelberg, Germany). The data were analyzed using the software FlowJo 10.4.2 (LLC, Asgland, OR, U.S.A.) and calculated as percentage of total cells. 2.10. Statistical Analysis. Data are presented as mean ± standard deviation. The statistical analysis was performed by one-way ANOVA followed by Tukey-Kramer test or Dunn’s test (nonparametric analysis). For this, SigmaPlot software was used (version 13.0, Systat Software GmbH; Erkrath, Germany). Values were considered as significantly different if p value < 0.05.

3. RESULTS 3.1. C. vulgaris Preparations Did Not Impair Cell Viability but Induced DNA Damage in Caco-2 and HT29 Cells. The applied doses of C. vulgaris were selected in order to clearly detect DNA strand breaks without impairing the cell viability. None of the used C. vulgaris preparations and control treatments resulted in a cell viability below 95% in both cell lines (data not shown). To examine potential cell specific effects, C. vulgaris-induced genotoxicity was tested in Caco-2 as well as in HT-29 cells by applying the comet assay. In Caco-2 cells, H2O2 (positive control) significantly induced DNA strand breaks. Likewise, the treatment with the higher concentration of C. vulgaris (200 μg dm/mL) resulted in a significant induction of DNA strand breaks. However, when C

DOI: 10.1021/acs.jafc.9b03457 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry applying C. vulgaris in a ten times lower concentration, the extent of DNA damage was diminished. Moreover, the results revealed no further adverse effect due to the application of sonication or ball-milling (Table 1). Table 1. Induction of DNA Strand Breaks by C. vulgaris Preparations in Caco-2 and HT-29 Cellsx Treatment Control (HBSS) H2O2 (100 μM) Unprocessed

Sonicated

Ball-milled

200 μg dm/ mL 20 μg dm/ mL 200 μg dm/ mL 20 μg dm/ mL 200 μg dm/ mL 20 μg dm/ mL

Caco-2 cells Tail intensity (%)

HT-29 cells Tail intensity (%)

4.40 ± 0.63a

4.38 ± 0.43a

23.2 ± 6.0b

13.7 ± 1.63b

24.6 ± 4.66b

16.6 ± 0.87c

9.18 ± 2.72ab

9.09 ± 2.12d

28.9 ± 3.52b

21.8 ± 1.68c

7.96 ± 0.55ab

10.3 ± 1.97d

30.8 ± 4.30b

8.93 ± 2.55ab

11.2 ± 3.53ab

4.67 ± 0.35a

Data are means of at least three independent experiments ± standard deviation. Different letters indicate statistically significant differences as determined separately for each processing method by one-way ANOVA on ranks followed by the Dunn’s test (p < 0.05). x

The data of HT-29 cells indicate that the extent of DNA damage induced by H2O2 and some C. vulgaris preparations were slightly lower than observed for Caco-2 cells. In contrast to the treatment with the ball-milled biomass (200 μg dm/ mL), the use of unprocessed as well as the sonicated biomass resulted in a significant induction of DNA damage. Also in HT-29 cells, the use of a ten times lower concentration showed a decreased genotoxic effect. However, merely the addition of ball-milled biomass at a concentration of 20 μg dm/mL led to no significant difference compared to the control treatment (Table 1). In order to identify a trigger for the genotoxic effect in Caco2 cells, fractionation of C. vulgaris suspension with regard to solubility in different solvents (Folch extraction) was performed. Data indicate that the fraction containing solid compounds significantly caused DNA damage. The hydrophilic and lipophilic subfractions only showed a slightly increased genotoxic effect (Figure 2A); however, water or lipid soluble peroxides could not be determined therein when applying the FOX-assay (data not shown). In addition, a C. vulgaris suspension was centrifuged in order to separate whole cells and cell wall fragments as well as cell organelles. Figure 2B shows that DNA damage was mediated mostly by the subfraction containing whole algal cells and bigger fragments, e.g., cell wall debris. 3.2. Processing lowered the amount of lipid peroxidation products (TBARS) in C. vulgaris preparations. Malondialdehyde, a byproduct of lipid peroxidation, is highly reactive, e.g., to form cross-links with DNA and proteins.34−36 The unprocessed C. vulgaris biomass revealed the highest amount of lipid peroxidation products (TBARs). Sonication and ball-milling of biomass significantly lowered the content (Table 2).

Figure 2. Induction of DNA strand breaks by several C. vulgaris fractions obtained by (A) Folch extraction and (B) centrifugation in Caco-2. (C) Control treatment. THF: tetrahydrofuran; P1: intact cells, cell, debris; P2: cell organelles; S2: cytosol; data are means of at least three independent experiments ± standard deviation. Different letters indicate statistically significant differences as determined by one-way ANOVA followed by Tukey-Kramer test (p < 0.05).

Table 2. Content of TBARs (Malondialdehyde) in C. vulgaris Preparationsx Treatment

Malondialdehyde nmol/mg dm

Unprocessed Sonicated Ball-milled

9.39 ± 0.26a 7.77 ± 0.19b 3.75 ± 0.24c

x Data are means of four independent measurements ± standard deviation. Different letters indicate statistically significant differences as determined by one way ANOVA on ranks followed by the TukeyKramer test (p < 0.05).

3.3. C. vulgaris Led to a Dose-Dependent ROS Formation in Caco-2 and HT-29 Cells. Figure 3A,B shows the formation of ROS, as a marker for induction of oxidative stress in Caco-2 and HT-29 cells after exposure to various C. vulgaris preparations using DCFH-DA as a fluorescence probe. H2O2 as well as TBHP were applied as positive controls, which significantly induced the formation of cellular ROS. The treatment with unprocessed C. vulgaris at a concentration of 200 μg dm/mL resulted in a significant formation of intracellular ROS in both cell lines. The use of processed C. vulgaris at the highest concentration led to a weaker level of cellular ROS. In general, all preparations D

DOI: 10.1021/acs.jafc.9b03457 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Formation of cellular reactive oxygen species (ROS) by preparations of undigested C. vulgaris (A) in Caco-2 and (B) HT-29 cells. (C) Control treatment. TBHP: tert-butylhydroperoxide; data are means of at least three independent experiments ± standard deviation. Different letters indicate statistically significant differences as determined separately for each processing method by one-way ANOVA on ranks followed by the Dunn’s test (p < 0.05).

Figure 4. Effect of recovery on (A) repair of DNA strand breaks and (B) proliferation after treatment with C. vulgaris (200 μg dm/mL) in Caco-2 cells. (C) Control treatment. Data are means of three independent experiments ± standard deviation. Different letters indicate statistically significant differences as determined separately for H2O2 and C. vulgaris by one-way ANOVA in part A on ranks followed by the Dunn’s test (p < 0.05) for H2O2 or by Tukey-Kramer test (p < 0.05) for C. vulgaris, and in part B by Tukey-Kramer test (p < 0.05).

showed a dose-dependent induction of oxidative stress in both cell lines. 3.4. C. vulgaris-Induced DNA Damage Is Temporary and Did Not Impair Cell Proliferation of Caco-2 Cells. The time-dependent repair of DNA damage induced by H2O2 as well as unprocessed C. vulgaris (200 μg dm/mL) and the effects on cell viability/proliferation were investigated in Caco2 cells. Figure 4A,B indicates that H2O2-induced DNA damage was not completely repaired after a 2 h recovery and the metabolic activity (viability) was significantly impaired, whereas the DNA strand breaks induced by C. vulgaris already seemed diminished after a recovery period of 2 h. However, even after 24 h of recovery, the tail intensity remained significantly increased in comparison to the control-treated cells. There was no effect on cell proliferation either for H2O2 or for C. vulgaris treated cells. 3.5. C. vulgaris Preparations Did Not Show an Apoptotic and Necrotic Response in Caco-2 and HT29 Cells. The induction of apoptosis was assessed by determining the activation of intracellular caspase-3 and the translocation of phosphatidylserine (Annexin V-staining). Moreover, necrotic cells were detected by using the Zombie Violet dye. Several positive controls and incubation times were tested to assess the optimal conditions showing an apoptotic effect (data not shown). The applied cell lines showed a heterogeneity in their sensitivity to the applied positive controls as well as the extent of positive effects in the applied tests. Therefore, incubation times of 48 h for undifferentiated

Caco-2 cells, 4 h for differentiated Caco-2 cells, and 24 h for HT-29 cells were chosen based on the activity of the respective positive controls. Neither 50 mM butyrate (positive control) nor the C. vulgaris preparations led to an increase of Annexin V-staining in Caco-2 cells. However, the incubation with butyrate significantly activated caspase-3. The application of the C. vulgaris preparations (20 mg dm/mL) did not exhibit an effect on caspase-3 activation or necrosis induction (Table 3). There was no apoptotic effect due to any treatment in differentiated Caco-2 cells when determining Annexin-V positive stained cells. However, the incubation with staurosporin (0.5 μM)/brefeldin A (0.5 μM) showed a significant increase of activated caspase-3. Moreover, neither the treatment with C. vulgaris preparations nor with negative controls (EMEM, ethanol) resulted in an increase of intracellular activated caspase-3 or a necrotic effect (Table 4). In HT-29 cells, the treatment with staurosporin (0.5 μM)/ brefeldin A (0.5 μM) led to a significant increase of Annexin Vpositive stained cells and caspase-3 activation. None of the C. vulgaris preparations showed an apoptotic effect in any of the applied methods. In addition, there was no induction of necrosis, though the number of positive stained (Zombie Violet dye) cells after staurosporin/brefeldin A treatment was E

DOI: 10.1021/acs.jafc.9b03457 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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similarities with intestinal cells due to differentiation of the confluent monolayer.37,38 This would represent an optimal model; however, the determination of DNA damage in differentiated cells is technically difficult. The undifferentiated cells are a more convenient and reliable model and were therefore applied for the depicted experiments. However, differentiated Caco-2 cells were used to test an apoptotic or necrotic effect of the test preparations. As already mentioned, the composition of C. vulgaris constituents can vary due to cultivation conditions, e.g., light intensity and composition of culture medium.4−7,39,40 The culture conditions in the present manuscript were selected on the basis of previous findings. TAP acetate-free medium is suitable for investigating photoautotrophic growth and was already tested successfully on a pilot scale using C. vulgaris.41,42 The optimal conditions for temperature (25.44 °C) and photon flux density (160 μmol m−2 s−1) are based on studies of C. vulgaris growth kinetics43.44 In commercial preparations, photoautotrophic cultivations of microalgae are usually performed outdoors. This means that the cultivation systems usually show environmentally based deviations in temperature and light availability due to day/night cycles.41,42 Hence, the culture conditions used in our work are only partially related to commercial cultivations. We need to mention that the biomass production in photobioreactor systems is usually higher compared to that in shake flask systems. To our knowledge, currently available C. vulgaris food products in the market only use whole cells and no single cell fractions. Usually, the wet and concentrated biomass is spray-dried and the respective powder is directly sold or added as supplementation to products such as beverages, noodles, pills, etc. Since the current food market mainly uses C. vulgaris as a whole cell product, the test preparations in this study are comparable to industrial products. The use of different processing methods proved to be necessary in order to enable a proper release and bioavailability of C. vulgaris nutrients.26 Sonication as well as ball-milling are common methods used for cell disruption, e.g., in food technology45 and processing of microalgae biomass.46 During sonication, radicals/oxidants might be formed45 which pose a risk for the induction of DNA damage. In the milling process, an increase of secondary lipid peroxidation products has also been observed.47 However, the depicted data reveal a decrease of lipid peroxidation products due to processing, which might be explained by (1) inactivation of necessary enzymes at the same time and (2) higher activity of antioxidants due to the release from cellular bindings. During processing by sonication and ball-milling, byproducts might be formed causing a deviating cellular effect. The treatment with the C. vulgaris preparations (unprocessed and processed) resulted in a dose-dependent induction of DNA strand breaks in Caco-2 as well as HT-29 cells. The ball-milled biomass showed the weakest tail intensity in HT-29 cells, whereas such an effect could not be observed in Caco-2 cells. Obtained data reveal that none of the applied processing methods led to a further increase of DNA strand breaks. Therefore, the applied conditions for sonication as well as for ball-milling might be assessed as safe for the processing of C. vulgaris. Safety hazards surrounding the consumption of Chlorella are rarely investigated, even though a potential for adverse health effects8,9,48 and information on relevant toxicology parameters have already been indicated.10,11 The used C. vulgaris biomass was cultivated under controlled laboratory conditions, thus contaminations with pathogenic microorganisms as reported

Table 3. Apoptosis Rate (Annexin V), Caspase-3 Activation, and Necrosis Rate of Undifferentiated Caco-2 Cells after Treatment with Controls and C. vulgaris Preparations (20 μg dm/mL)x Treatment Control (EMEM) Butyrate Unprocessed Sonicated Ball-milled

Annexin V (%) 3.43 4.99 3.35 3.31 3.82

± ± ± ± ±

0.98 0.98 0.49 1.76 2.24

Caspase-3 activation (%) 2.26 14.2 2.28 2.12 2.02

± ± ± ± ±

0.69y 4.08b 0.79b 0.63b 0.71

Necrosis (%) 4.59 6.91 5.68 7.31 7.03

± ± ± ± ±

1.88 3.48 3.66 4.24 3.93

Data are means of four independent experiments ± standard deviation. Different letters indicate statistically significant differences as determined by one-way ANOVA on ranks followed by the Dunn’s test (p < 0.05). yButyrate vs control; p = 0.052. x

Table 4. Apoptosis Rate (Annexin V), Caspase-3 Activation, and Necrosis Rate of Differentiated Caco-2 Cells after Treatment with Controls and C. vulgaris Preparations (20 μg dm/mL)x Treatment Control (EMEM) Ethanol Staurosporin/ Brefeldin A Unprocessed Sonicated Ball-milled

Annexin V (%)

Caspase-3 activation (%)

Necrosis (%)

2.76 ± 1.60 3.97 ± 1.59 1.95 ± 0.54

3.75 ± 1.58ay 3.92 ± 0.33b 14.56 ± 5.43b

11.17 ± 5.46 10.67 ± 6.20 9.75 ± 3.52

2.57 ± 0.63 3.00 ± 0.90 4.44 ± 2.17

2.99 ± 1.26 2.74 ± 0.46 3.50 ± 2.28b

7.71 ± 3.20 7.69 ± 3.19 9.33 ± 3.97

x Data are means of four independent experiments ± standard deviation. Different letters indicate statistically significant differences as determined by one-way ANOVA on ranks followed by the Dunn’s test (p < 0.05). yStaurosporin/Brefeldin A vs control; p = 0.051.

significantly higher compared to the incubation with C. vulgaris preparations (Table 5). Table 5. Apoptosis Rate (Annexin V), Caspase-3 Activation, and Necrosis Rate of HT-29 Cells after Treatment with Controls and C. vulgaris Preparations (20 μg dm/mL)x Treatment Control (DMEM) Ethanol Staurosporin/ Brefeldin A Unprocessed Sonicated Ball-milled

Annexin V (%)

Caspase-3 activation (%)

Necrosis (%)

2.98 ± 0.93ay 2.52 ± 0.70 16.1 ± 6.12b

0.45 ± 0.16 0.39 ± 0.07 16.6 ± 7.49b

2.63 ± 1.44b 2.76 ± 1.70b 7.20 ± 4.40b

2.90 ± 0.59b 2.83 ± 1.13ay 4.09 ± 3.72b

0.36 ± 0.08 0.39 ± 0.08 0.41 ± 0.09b

1.69 ± 0.81 1.84 ± 1.33 1.89 ± 1.48

Data are means of four independent experiments ± standard deviation. Different letters indicate statistically significant differences as determined by one-way ANOVA on ranks followed by the Dunn’s test (p < 0.05). yStaurosporin/Brefeldin A vs control (p = 0.059); Staurosporin/Brefeldin A vs sonicated C. vulgaris (p = 0.057). x

4. DISCUSSION The present study investigated the cytotoxicity, genotoxicity, and induction of apoptosis of different C. vulgaris preparations in undifferentiated Caco-2 and HT-29 cells. These cell lines originated from the colon adenom (Caco-2) or the carcinom (HT-29). They show morphological and biochemical F

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the diets were well tolerated and there were no adverse or toxic effects.10,12,15 Moreover, Janczyk et al. performed a threegeneration reproduction study in mice and could not observe any adverse effects.13 To perform a more detailed investigation of the genotoxic potential of C. vulgaris, the application of more doses and variation of incubation times might be required, i.e., in accordance with the necessary guidelines. However, the scope of the depicted experiments was to provide an initial clue regarding the genotoxicity of C. vulgaris depending on the use of processing methods in human colon epithelial cells. There is a hint that the induction of DNA damage might be related to cell wall components and the formation of ROS. However, it was observed that in the case of the preparations of ball-milled C. vulgaris, the genotoxic effect differs when comparing Caco-2 and HT-29 cells. This cell-type-specific effect might be explained by the compositions of their outer cell membrane (e.g., mucin production). In conclusion, the data indicate that C. vulgaris exhibits a genotoxic potential in a dose-dependent manner; although, the induced DNA strand breaks were already repaired after a recovery time of 2 h. Mainly, cell wall components trigger the genotoxic effect. However, a longtime incubation revealed that there was no impairment of cell proliferation or induction of apoptosis. No genotoxic effects or further adverse effects were related to processing by sonication or ball-milling.

for cultivation in open ponds can be disregarded. We assumed that a contribution of H2O2 or the further generation of radicals and lipid peroxidation products caused DNA damage. However, a coincubation with catalase revealed no differences in the C. vulgaris-induced DNA strand breaks (data not shown). Eventually, a high level of H2O2 in C. vulgaris preparations or its formation during the incubation, as shown for several substances, e.g., phenolic compounds in different cell culture media, was also disregarded.49,50 The induction of DNA damage and the apoptotic signaling cascade in HepG2 cells were observed following the treatment with a C. vulgaris hot water extract.11 Consequently, the application of various C. vulgaris extracts or fractions might help to identify the trigger for DNA strand breaks. The depicted data reveal a significant induction of DNA damage caused by a solid subfraction (Folch extraction). Moreover, the lipophilic as well as the hydrophilic subfractions showed a genotoxic potential; however, it was four times less than that observed for the solid fraction. Due to the results of the FOXassay, the presence of peroxides in the latter fractions might be excluded. Likewise, the application of a subfraction (obtained by centrifugation) composed of intact cells, cell debris, and the cell wall resulted in a significant induction of DNA damage. It might be assumed that components of the C. vulgaris cell wall such as the extracellular bounded groups as well as the surface structure caused the genotoxic effect by interaction with the cell membrane. This leads to the suggestion that their removal might decrease the genotoxic potential. For application in food products and nutraceuticals, lipid and protein extracts of Chlorella were of interest, since they are commonly used for other microalgae species. Therefore, it might be of interest that a lipophilic and a hydrophilic subfraction showed a significantly lower extent of DNA damage. So far, the C. vulgaris-induced DNA damage could not be attributed to one single compound, it might be due to a multitude of compounds related to the C. vulgaris cell wall or surface. The depicted data indicate a dose-dependent formation of ROS in both cell lines. Here, the use of unprocessed biomass led to a huge amount of cellular ROS; however, when using the processed biomass, the ROSconcentration was about 3−5 times lower. The underlying mechanisms of the observed effects are presently unclear. However, the induction of oxidative stress might be one explanation for the noted DNA damage. It remains uncertain if the presence of specific cell wall components or if the interaction with the C. vulgaris cell surface causes the genotoxic effects. Moreover, processing did not successfully impair the activity of the trigger of the genotoxic effect. In contrast to the H2O2-induced DNA damage, that caused by 200 μg C. vulgaris dm/mL was already repaired after a recovery time of about 2 h. Neither the Chlorella preparations nor H2O2 indicated an adverse effect on cell proliferation. In addition, a rapid repair of single strand breaks has also been reported for substances with a high genotoxic activity in different cell lines.51,52 Moreover, the depicted data reveal no induction of apoptosis in Caco-2 (undifferentiated and differentiated) or HT-29 cells due to any of the applied preparations. In contrast, Cha et al. reported an apoptotic effect of ethanolic Chlorella extracts in colon cells.53 However, previous studies also failed to find a hint of toxicity, mutagenicity, or any other adverse effects when testing Chlorella protothecoides flour in in vitro and in vivo trials.10,14,54 Several Chlorella species were investigated in vivo and added as supplements to the diets of rodents. All studies confirmed that

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 7216625349. Fax: +49 7216625404. E-mail: [email protected]. ORCID

Andrea Gille: 0000-0002-3992-1099 Funding

This work by Andrea Gille was supported by a grant (7533− 10−5/91/2) from the Ministry of Science, Research and the Arts of Baden-Württemberg, Germany as part of the BBW ForWerts Graduate Program. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully thank Selina Chabauty, Dr. Meike Dössel, Anny Krämer, Renate Lambertz, Barbara Mathony-Holschuh, Konstanze Schelm, and Lars Uhlmann for their contribution and excellent technical assistance during the performance of the experiments.

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REFERENCES

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