Inflammatory Responses and Potencies of Various

Toxicon 97 (2015) 23e31

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Toxicon

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Inﬂammatory responses and potencies of various lipopolysaccharides from bacteria and cyanobacteria in aquatic environments and water supply systems

* Yumiko Ohkouchi a, , Satoshi Tajima b, 1, Masahiro Nomura b, 2, Sadahiko Itoh b a Department of Global Ecology, Graduate School of Global Environmental Studies, Kyoto University, Kyoto Daigaku-Katsura CI-2-233, Nishikyo-ku, Kyoto, 615-8540, Japan b Department of Environmental Engineering, Graduate School of Engineering, Kyoto University, Kyoto Daigaku-Katsura CI-2-233, Nishikyo-ku, Kyoto, 615-8540, Japan article info abstract

Article history: Inflammatory substances derived from indigenous bacteria in aquatic environments or water systems are Received 5 September 2014 of great concern. Lipopolysaccharides (LPSs), one of the major inflammatory substances in water, are Received in revised form usually identified using Limurus amoebocyte lysate (LAL) assay on the basis of their endotoxic activity, 5 January 2015 but endotoxin levels do not accurately represent their inflammatory potency in humans. In this inves- Accepted 5 February 2015 tigation, the cellular endotoxin contents of pure-cultured bacteria/cyanobacteria, which are frequently Available online 7 February 2015 detected in water sources and distribution systems, and of indigenous bacteria in a river and in biologically activated carbon (BAC) effluent, were investigated. The indigenous bacteria showed the highest Keywords: 3 fi Lipopolysaccharide (LPS) endotoxin contents exceeding 10 EU/cell. The LPSs were then puri ed from those samples, and their fl Purification in ammatory potencies were examined using a human monocytic cell line. The LPSs from Acinetobacter Endotoxin lwoffii culture, the river water, and the BAC effluent sample revealed a unique cytokine secretion pattern; Indigenous bacteria/cyanobacteria they induced both IL-8 and TNF-a more strongly than the other tested bacterial LPSs. These results Human monocytic cells suggest that natural bacterial/cyanobacterial flora in aquatic environments and water distribution sys- Cytokine secretion tems have the potential to induce relatively strong inflammatory responses in humans; therefore, further accumulation of data on water quality from the perspective of not just endotoxins but inflammatory potency is needed. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction cell surface. LPSs are also called endotoxins because of their biological activity. Information on endotoxin levels in various water There are various naturally occurring substances that are toxic samples has been accumulated. Just as an example, drinking water to humans in aquatic environments. Cyanobacterial toxins, such as in Japan, China, and Australia ranges 1e10, 4e10, and 72e186 EU/ microcystins, are one of the major toxic compounds in aquatic mL, respectively, while the endotoxin levels in source water varied environments (Carmichael and Bent, 1981). Lipopolysaccharides largely, but normally are in the range of 10e1000 EU/mL (Ohkouchi (LPSs), which are the outer membranes of gram-negative bacterial et al., 2007, 2009; Can et al., 2013; O'Toole et al., 2008). Rapala et al. or cyanobacterial cells, are also widely known to cause strong (2002) also reported that water bloom sample with dense cyano- innate immune reactions in humans via Toll-like receptors on the bacteria reached at 3.8 104 EU/mL in Finland. Limurus amoebocyte lysate (LAL) assay is widely used to determine endotoxin levels in aquatic environments. This assay, based on coagulating reactions * Corresponding author. Present address: School of Life and Environmental Sci- of LAL with endotoxins, is very sensitive and can detect small ence, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa, amounts of endotoxins. In this assay, the endotoxic activity of each 252-5201, Japan. bacterial LPS was expressed in terms of its endotoxic equivalence E-mail address: [email protected] (Y. Ohkouchi). 1 Present address: West Japan Railway Company, Shin-Kokusai Building 9F, 4e1, with the LPS of Escherichia coli-type strains. However, several Marunouchi 3-chome, Chiyoda-ku, Tokyo, 100-0005, Japan. studies have implied that there are big differences between the 2 Present address: Earthtech Toyo Co., Ltd., 44-32 Nishioji-cho, Daigo, Fushimi- endotoxic activities of various bacteria determined by LAL assay ku, Kyoto, 601-1374, Japan. http://dx.doi.org/10.1016/j.toxicon.2015.02.003 0041-0101/© 2015 Elsevier Ltd. All rights reserved. 24 Y. Ohkouchi et al. / Toxicon 97 (2015) 23e31 and their toxic effects on human health (Hansen et al., 1999; Dehus would help in the development of a novel strategy of water quality et al., 2006). Our previous study indicated that the cellular assay management. system using a monocytic cell line, THP-1, could be useful for This investigation focused on several bacterial or cyanobacterial detecting the changes in inflammatory potencies caused by bac- strains which ubiquitously inhabit water sources or distribution terial communities in aquatic environments (Ohkouchi et al., 2012). systems to determine what kind of bacteria could contribute to an However, the information on their inflammatory potencies as increase in inflammatory potencies as well as endotoxins in water caused by indigenous bacteria in various water samples is still supply systems. Firstly, the cellular LPS contents of each pure insufficient, as is that on their endotoxic activities. cultured bacteria/cyanobacteria or indigenous bacteria in river In water supply systems, there are three sites where endotoxins water or BAC effluent were compared. Secondly, their endotoxic can increase: at water sources as previously described (Rapala et al., activities and the inflammatory potencies of LPSs after purification 2002), in a treatment process using biologically activated carbon were also compared using a monocytic cell line based on the levels (BAC) (Ohkouchi et al., 2007; Can et al., 2013), and in distribution and patterns of cytokine secretion to narrow down which strains systems (Ohkouchi et al., 2009). In general, cyanobacteria are can impact human health. Also, a possibility that contaminant recognized as major contributors to the increase of endotoxins in substances contribute to inflammatory potencies by the purified water sources, while gram-negative bacteria could be a dominant LPSs pretreated with/without polymixin B was examined. Lastly, factor in treatment processes and distribution systems. Ohkouchi the importance of water quality in terms of inflammatory potency et al. (2007) compared the endotoxic activities derived from was discussed based on the data obtained. Microcystis aeruginosa, Synechococcus sp. and E. coli cultured in the laboratory. The results showed that the Synechococcus sp. could be a 2. Materials and methods major contributor to endotoxin increase in the Lake Biwa - Yodo River Basin due to their abundance in the natural aquatic envi- 2.1. Bacterial/cyanobacterial culture conditions ronment, especially in summer. The survey results also suggested that effluents from sewage treatment plants could be sources of The bacteria/cyanobacteria used for the LPS preparation and endotoxin contamination other than cyanobacterial bloom in that their cultivation conditions are listed in Table 1. These bacteria/ basin. The second site for the increase of endotoxins could be BAC cyanobacteria were chosen based on published data regarding their processes, which provide habitats for bacteria. Bacteria can occurrence rates in water sources or distribution systems. multiply on granular activated carbon (GAC) by using nutrients in Microcystis aeruginosa and Synechococcus sp., which are frequently water. The proteobacteria, i.e. gram-negative bacteria, were found to detected in water sources (Rapala et al., 2002; Hoson et al., 2002), be dominant inhabitants in the bacterial community created on the were cultured at 20 C for 20e30 days with illumination of surface of BAC (Poitelon et al., 2010). In contrast, it is known that 1500e2500 lux at 12-h intervals using CB and C media, respec- the bacterial populations in water distribution systems vary from tively. Gram-negative bacteria, except Escherichia coli NBRC 3301, system to system depending on their conditions. In many cases, the which are frequently detected in tap water samples or in biofilm bacteria in biofilm accumulated inside the pipes or in tap water accumulated in distribution systems, were cultured at 20 C for samples have been detected using culture techniques, but some 4e12 days using a medium recommended by each culture collec- researchers have applied DNA-based molecular techniques. Various tion. E. coli NBRC 3301 was cultured at 37 C for 30 h using LB species of gram-negative bacteria that belong to alpha-, beta-, and medium. gamma-proteobacteria were frequently detected. For example, Hirata et al. (1993) detected Alcaligenes sp., Bacillus sp., Methyl- 2.2. Water sampling obacterium sp., Pseudomonas sp., and Flavobacterium sp. in distrib- uted water samples. Furuhata et al. (Furuhata, 2004) also reported A 21.8 L volume of river water was taken from the Yodo River that Methylobacterium sp. and Pseudomonas sp. were dominant downstream of Osaka (N34.72.48.47, E135.51.30.37) in December species in water samples taken at a hospital via a storage tank. Scott 2010. The sampling site was adjacent to the intake area of a fi and Pepper (2010) identi ed frequently detected bacterial species drinking water purification plant. A 20 L volume of effluent water in tap water samples in the United States as Sphingomonas sp., from the BAC adsorption process was taken at the water purifica- Acidovorax sp., Aquabacterium sp., and Acinetobacter sp. based on tion plant on the Yodo River. The purification plant has coagulation- the homology of 16S rRNA sequences. The bacterial regrowth of sedimentation, mid-ozonation, rapid sand filtration, post- these various species, except for Sphingomonas sp., whose mem- ozonation, BAC, and chlorination processes. The containers used brane structure differs from typical LPSs (Kawasaki et al., 1994; for water samplings were soaked with PyroCLEAN (Alerchek, Inc., Kawahara et al., 1999), could cause increases in endotoxin and in- Portland, ME, USA) for more than one hour to degrade and solu- fl ammatory potency during water distribution. Therefore, identi- bilize endotoxins attached on the inner surface and rinsed thor- fying and prioritizing the major contributors to increases in oughly with endotoxin-free Milli-Q water. The endotoxin-free fl endotoxin and in ammatory potency in water supply systems Milli-Q water was produced using a Milli-Q Academic equipped

Table 1 Bacterial strains tested in this investigation.

Tested bacteria Strain Class Proliferation site Culture condition Reference

Escherichia coli NBRC 3301 g-Proteobacteria e LB broth, 37 C, 30 h Microcystis aeruginosa NIES-44 Cyanophyceae Water resources CB medium, 20 C, 30 days (Rapala et al., 2002) Synechococcus sp. NIES-946 Cyanophyceae Water resources C medium, 20 C, 20 days (Hoson et al., 2002) Pseudomonas fluorescens ATCC 49642 g-Proteobacteria Distribution systems R2A broth, 20 C, 5 days (Yano et al., 2009) Pseudomonas aeruginosa NBRC 12689 g-Proteobacteria Distribution systems NBRC 802, 20 C, 12 days (Yano et al., 2009)(Szita et al., 2007) Aquabacterium commune ATCC BAA-209 b-Proteobacteria Distribution systems ATTC 2261, 20 C, 7 days (Kalmbach et al., 1999) Acidovorax delafieldii ATCC 17606 b-Proteobacteria Distribution systems Nutrient broth, 20 C, 4 days (Lee et al., 2010) Acinetobacter lwoffii JCM 6840 g-Proteobacteria Distribution systems Nutrient broth, 20 C, 7 days (Scott and Pepper, 2010) Methylobacterium fujisawaense NBRC 16843 a-Proteobacteria Distribution systems NBRC 352, 20 C, 7 days (Furuhata et al., 2006) Y. Ohkouchi et al. / Toxicon 97 (2015) 23e31 25 with a BioPak cartridge as a final filter. The water sample was (EU). Each sample was diluted with endotoxin-free Milli-Q water. transported to our laboratory under refrigerated conditions at 4 C. Pipette chips and 96-well microplates guaranteed to be endotoxin- free were used for the assay. 2.3. Enumeration of bacterial cells For determination of endotoxin in pure culture, the culture media samples were taken at late logarithmic growth phases. The The bacterial cell counts cultured in the laboratory were deter- endotoxins of media themselves for cultivation in Table 1 were also mined by pour-plating using agar plates under the conditions determined before inoculation. The known weight of the purified recommended by each culture collection. The Microcystis cell count LPS samples were dissolved in sterilized endotoxin-free Milli Q was determined by microscopic examination at x400 magnification water, and then the endotoxins were also determined in the same after pressurization. The Synechococcus cell count was determined way. by epifluorescence microscopy observation of intrinsic red fluo- rescence under G-excitation. The culturable heterotrophic bacteria 2.6. Cell culture in the river and BAC effluent water samples were enumerated using R2A agar plates at 20 C with 7 days incubation. The total bacterial A monocytic cell line, THP-1 (JCRB0112.1), purchased from the cell counts were determined by epifluorescence microscopic Japanese Collection of Research Bioresources Cell Bank, was enumeration under UV excitation after 40,6-diamidino-2- cultured in RPMI1640 medium with 5% heat-inactivated FBS. All phenylindole (DAPI) staining. culture flasks were incubated at 37 C in humidified 5% CO2.

2.4. LPS puriﬁcation 2.7. Endotoxin stimulation

The bacterial cells and Synechococcus sp. cultured in our labo- The non-adherent THP-1 cells were suspended in each medium ratory were harvested by centrifugation at 8000 rpm for 10e20 min at 1.0 106 cells/mL. Aliquots of 100 mL cell suspensions were and then washed three times with sterilized 50 mM phosphate seeded in flat-bottomed 96-well culture plates and were incubated buffer (pH 7.2). Only Microcystis aeruginosa was harvested by for three hours prior to LPS stimulation. The commercial LPS of centrifugation at 6000 g for 10 min and then washed three times E. coli O55:B5 (Sigma Aldrich Japan, Tokyo, japan) and the extracted with sterilized 15 mg/L NaHCO3 solution. The Yodo River sample LPS samples were dissolved in endotoxin-free Milli-Q water and and the BAC effluent sample were filtered through a glass fiber filter filtered through a sterilized 0.2-mm syringe filter (DISMIC-25CS, (Advantec Toyo Kaisha, Ltd., GA-100, Tokyo, Japan) and then Advantec Toyo Kaisha, Ltd., Tokyo, Japan). After preincubation, the enriched using an ultrafiltration device with membranes of MWCO culture medium in each well was removed and 100 mL of fresh 10,000 (Advantec Toyo Kaisha, Ltd., Q0100, Tokyo, Japan). The de- media was added. The THP-1 cells were stimulated with LPS sam- vices and membranes were washed with 5 N and 1 N NaOH, ples for six hours by adding 10 mL LPS suspension (Ohkouchi et al., respectively, by soaking for more than three hours to degrade 2012). contaminated organics, and then rinsed thoroughly with endotoxin-free Milli-Q water. 2.8. Contaminant assay by blocking endotoxin activity After the cell pellets or the enriched water sample was lyophilized, LPSs were extracted from the lyophilized cells according to Polymixin B, cationic polypeptide, can bind to mono or the method reported by Bernardova et al. (2008). A brief descrip- diphosphate group of the lipid A (Coyne and Fenwick, 1993), and so tion of the procedure follows. An equal volume of hot 90% phenol- it is often used to neutralize biological activity of LPSs. The inhibi- water mixture was added to the suspension of lyophilized cells in tion of cytokine secretions by addition of polymixin B can exclude endotoxin-free water, and then stirred at 65e70 C for 20 min. This the possibility that contaminants remaining in the purified samples extraction procedure was repeated twice. After centrifugation, the contribute to cytokine production. The E. coli O55:B5 LPS (Sigma- supernatant aqueous phase was collected and dialyzed using eAldrich Japan), the LPSs extracted from Acinetobacter lwoffii JCM Spectra/Por 7 (1000 Da; Spectrum Laboratories, Inc., Rancho 6840, the river water, and the BAC effluent samples (5000 EU/mL) Dominguez, CA, USA) against endotoxin-free Milli-Q water for 3e4 were pretreated with 50 mg/mL polymixin B sulphate (Sigma days. The dialyzed sample was lyophilized again, and dissolved in Aldrich Japan) for 1 h at room temperature. Then, THP-1 was 0.1 M TriseHCl buffer containing 25 mg/mL RNase A (from bovine stimulated with the pretreated LPSs at 500 EU/mL in the same way pancreas, Waco Pure Chemicals, Osaka, Japan). After incubation for as described above. 16 h at 37 C to degrade RNA in the samples, an equal volume of 90% phenol-water solution was added again and stirred vigorously. The 2.9. Determination of cytokine levels and cell counts aqueous phase containing LPS was separated by centrifugation at 9600 g and dialyzed sufficiently against endotoxin-free water After stimulation with LPS samples, the culture supernatants using Amicon Ultra-15 (Millipore Japan, Tokyo). Finally, the dia- were collected and stored at 80 C until cytokine determination. lyzed sample was lyophilized and stored at 80 C until further An aliquot of 100 mL fresh medium was added to each well again, assay. and the cells were incubated for 30 min. Then, viable cell counts were determined using Cell counting Kit-8 (Dojin Laboratories, 2.5. Endotoxin determination Tokyo, Japan). The cytokines, TNF-a and IL-8, were measured by indirect sandwich ELISA assays (Diaclone, Gen-Probe Inc., San The endotoxins in pure bacterial/cyanobacterial culture, the Diego, CA, USA) following the manufacturer's instructions. water samples, and the purified LPSs were determined by kinetic- chromogenic LAL assay using Pyrochrome with Glucashield buffer 2.10. Statistical analysis (Seikagaku Biobusiness Corporation, Tokyo, Japan). The uninten- tional reaction with b-glucan remaining in the samples was blocked Data were compared by t-test using GraphPad Prism version 5.0 using Glucashield buffer. LPS purified from Escherichia coli strain (GraphPad Prism Inc., San Diego, CA, USA), and significant differ- O113:H10 (Seikagaku Biobusiness Corporation, Tokyo, Japan) was ences were determined with a level of p < 0.05. Principal compo- used for calibration, and the results were given in endotoxin units nent analysis was also applied to data set of 3 variables (cellular 26 Y. Ohkouchi et al. / Toxicon 97 (2015) 23e31 endotoxin contents, two types of cytokine secretions at LPS doses of However, the water purification plant where we conducted sam- 1000 EU/mL) obtained from 11 different LPS samples using the pling replaced a subset, not all, of the BAC columns with new GAC Microsoft Excel add-in software, Mac Tahen ver. 1.0a (ESUMI Co., every 5 years in order to attain constant organic removal. Therefore, Ltd., Tokyo, Japan). the effect of the operation period of BAC does not need to be taken into account in this case. The result also indicates that bacteria ubiquitously present in the water source or even after the water has 3. Results and discussion undergone drinking-water treatment have relatively high endotoxic activities. 3.1. Comparison of endotoxic activities derived from different Based on the comparison of the cellular endotoxin contents of bacterial strains and water samples each bacteria/cyanobacteria, there is a possibility that Aquabacterium commune, Pseudomonas spp., and The endotoxic activities of the pure bacteria/cyanobacteria Acinetobacter lwoffii, which exhibited endotoxin contents around strains cultured in our laboratory were determined before LPS 10 3 EU/cell, may be major contributors to endotoxin increases that extraction. The results in terms of endotoxins per cell in each cul- occur during water distribution. Two Pseudomonas spp. strains ture medium are compared in Fig. 1. The endotoxin in each culti- were frequently detected in biofilm samples or tap water samples vation medium was negligible less than 0.23 EU/mL. The endotoxic in many countries (Yano et al., 2009; Szita et al., 2007). Additionally, activities of indigenous bacteria in river water/BAC effluent samples Kalmbach et al. (1999) reported that Aquabacterium commune is a are also shown in Fig. 1. The endotoxic activities per cell were dominant and ubiquitous community member in biofilms accu- calculated by dividing the endotoxins determined by LAL assay by mulated in several distribution systems in Hamburg, Berlin, Mainz, the total cell counts in the samples determined by DAPI staining, and Stockholm, which had different water sources and treatment because LPSs on cell surfaces exhibit endotoxic activity regardless processes. Acinetobacter lwoffii, which is known as an opportunistic of the viability of bacterial cells. Aquabacterium commune and pathogen, was also widely detected in water supply systems (Scott Pseudomonas aeruginosa showed the highest endotoxic activities, and Pepper, 2010). If these ubiquitously present bacteria, which greater than 10 3 EU/cell. The endotoxic activities per cell of have potent endotoxic activity, multiply in water distribution sys- Acinetobacter lwoffii, Synechococcus sp., Pseudomonas fluorescens, tems, significant elevations of endotoxin levels in water could Microcystis aeruginosa, Acidovorax delafieldii, and Escherichia coli occur. followed in descending order. In contrast, significantly lower endotoxic activities were detected in Methylobacterium fujisa- 3.2. LPS purification from pure bacterial cultures, river water, and waense, less than 10 9 EU/cell. BAC effluent The endotoxin levels of the river water and the BAC effluent were found to be 440 and 13 EU/mL, respectively. The endotoxic Table 2 shows the changes in endotoxic activities and dry activity per cell of indigenous bacteria in the BAC effluent water weights during the LPS purification steps from the pure bacterial was slightly higher than that in the river water (river water cultures and the indigenous bacterial cells in the river water or the 1.1 10 3;BACeffluent 1.5 10 3 EU/cell). Both water samples BAC effluent samples. The results showed that the endotoxic ac- were taken in the winter season; therefore, gram-negative bacteria tivities per dry weight were not always increased by purification in rather than cyanobacteria were considered to be major contribu- the case of pure bacterial cultures. Theoretically, the total endotoxic tors to the endotoxic activities. This result showed that the cellular activities per dry weight should have increased as the purity endotoxin content was somewhat increased in the BAC effluent, increased due to the removal of many of the coexisting cellular whereas 97% of endotoxins in the river water regarded as source components. In fact, the dry weights after purification were largely water were removed through rapid sand filtration and ozonation- reduced by 0.8e10%. However, the total endotoxins were increased BAC processes. The increase can be attributed to the shift in the in some cases in pure culture samples after phenol extraction, bacterial community during treatment processes, especially the which destroys cell membranes and removes proteinaceous com- BAC adsorption process, which is known to be a place for bacterial pounds contained in bacterial cells by denaturing. In contrast, the regrowth. Kasuga et al. (2011) reported that the bacterial commu- endotoxic activities per unit weight of indigenous bacteria in water nity structures in the BAC can shift with the maturing process. samples were increased step-by-step during purification. The different behaviors of the endotoxic activities in the environmental samples from the cultured samples during purification might arise due to changes in the association state of the LPSs and in the damage to the cell membrane, or due to a non-specific LAL reaction with some components contained in the bacterial cultures, but the exact reason is still unclear. An additional data accumulation would be needed. The endotoxic activities per dry weight in the purified LPSs are compared in Fig. 2. Synechococcus sp. exhibited the highest endotoxic activity of 50 EU/ng, and the activities of Aquabacterium commune and Escherichia coli followed at levels greater than 10 EU/ ng. Generally, the endotoxic activities of purified LPSs from Escherichia coli were reported in the range of 5e10 EU/ng (Anderson et al., 2003; Bernardova et al., 2008). The endotoxic activity of the purified LPS from E. coli NBRC 3301 cultured in our laboratory was consistent with that value, and this consistency suggests that the purification procedure was adequate. The endotoxic activity of the LPS purified from the river water Fig. 1. Comparison of cellular endotoxin contents of pure-cultured bacteria/cyano- fi bacteria and water samples. Those values are calculated by dividing the endotoxic sample was comparable with the LPSs puri ed from two Pseudo- activity in the culture medium or water samples by the total cell counts. monas species. In contrast, the LPS purified from the BAC effluent Y. Ohkouchi et al. / Toxicon 97 (2015) 23e31 27

Table 2 Changes of endotoxic activities and dry weight during puriﬁcation steps.

Bacteria/water Harvested After phenol extraction Puriﬁed samples Endotoxin Dry Endotoxin per dry Endotoxin Dry Endotoxin per dry Endotoxin (EU) Dry weight Endotoxin per dry (EU) weight (mg) weight (EU/ng) (EU) weight (mg) weight (EU/ng) (mg) weight (EU/ng)

E. coli 4.1 107 280 1.48 10 1 7.4 108 29.6 2.49 101 3.8 107 2.5 1.51 101 M. aeruginosa 3.9 105 34.7 1.12 10 2 9.9 104 8.9 1.11 10 2 5.4 103 1.3 4.13 10 3 Synechococcus sp. 1.4 108 194 7.12 10 1 6.4 107 15.2 4.18 100 1.7 108 3.3 5.06 101 River water 9.6 106 20.3 4.72 10 1 4.8 106 4.4 1.09 100 2.2 106 1.7 1.29 100 P. fluorescens 1.9 109 516 3.67 100 2.6 108 104 2.49 100 3.0 107 13.3 2.27 100 P. aeruginosa 9.3 108 339 2.74 100 4.3 108 70.2 6.13 100 5.5 106 3.9 1.41 100 A. commune 6.2 108 106 5.84 100 6.9 108 16.3 4.23 101 3.2 107 1.4 2.29 101 A. delafieldii 2.0 107 266 7.51 10 2 1.6 108 37.0 4.32 100 1.0 105 2.3 4.35 10 2 A. lwoffii 1.4 108 111 1.26 100 5.2 108 14.3 3.64 101 9.5 105 1.5 6.32 10 1 M. fujisawaense 7.3 101 140 5.20 10 4 2.7 101 12.5 2.16 10 3 5.9 10 1 2.8 2.09 10 7 BAC effluent 4.3 105 6.0 7.13 10 2 4.5 105 1.4 3.24 10 2 3.4 104 0.6 5.73 10 2

sample showed much lower endotoxic activity, less than 0.1 EU/ng. Aquabacterium commune, and Acidovorax delafieldii exhibited few Also Microcystis aeruginosa LPS revealed much lower endotoxin IL-8 secretions over the entire endotoxin range. Regarding the two activity than E. coli LPS as reported by Blahov a et al. (Blahov a et al., Pseudomonas spp. and Aquabacterium commune, very slight in- 2013). creases in IL-8 secretion were observed at the extremely high endotoxin dose of 10000 EU/mL, but no significant doseeresponse relationship was observed. 3.3. Comparison of inflammatory responses caused by the purified As described previously, the TNF-a secretions were much lower LPSs than the IL-8 secretions in most bacterial samples other than Aci- netobacter lwoffii LPS. These results are consistent with previous The IL-8 and TNF-a secretions from THP-1 after six hours reports concluded that gram-negative bacteria more strongly stimulation with each purified LPS are presented in Fig. 3 and Fig. 4, induced IL-10-, IL-8-, and IL-6-type cytokines than gram-positive respectively. The cell counts of THP-1 after six hours stimulation bacteria, while gram-positive bacteria induced more of other showed no significant change in the examined range of LPSs puri- types of cytokines such as IFN-g, TNF-a, and IL-1b (Skovbjerg et al., fied from different lab-cultured bacteria/cyanobacteria. Each bac- 2010). However, only Acinetobacter lwoffii showed greater TNF-a terial/cyanobacterial LPS induced different potencies and patterns secretion in the examined endotoxin range. Erridge et al. (2007) of inflammatory responses in THP-1. Most LPSs induced higher IL-8 reported that Acinetobacter baumannii and Acinetobacter secretions than TNF-a, whereas only Acinetobacter lwoffii LPS ‘genomespecies 9’ LPSs showed high potency to induce IL-8 and induced TNF-a secretions that were twice as high as IL-8. Methyl- TNF-a in differentiated THP-1 cells. They indicated that stimulation obacterium fujisawaense LPS, which showed very little endotoxic with Acinetobacter LPSs induced significantly higher TNF-a secre- activity, induced IL-8 at 36 pg/106 cells, even at 0.2 EU/mL. Because tion even at 1 ng/mL LPS. They did not mention the levels of of its extremely low endotoxic activities, the LPS from Methyl- endotoxic activity of the Acinetobacter LPSs in that research, but obacterium fujisawaense was excluded from the following analyses. they explained that Acinetobacter baumannii LPS exhibited endo- The IL-8 secretion levels caused by the other LPSs were categorized toxic activity and cytokine secretions equal to those of Escherichia into three groups: the first group including Acinetobacter lwoffii and coli LPS. In our investigation, Figs. 3 and 4 clearly show that the Microcystis aeruginosa showed high levels of IL-8 secretion over purified A. lwoffii LPS induced 8- and 20-times higher secretions of 1000 pg/106 THP-1 cells at a dose of 1000 EU/mL. The second group IL-8 and TNF-a, respectively, than did E. coli at a dose of 1000 EU/ including Escherichia coli and Synechococcus sp. showed moderate mL, although A. lwoffii LPS had one-tenth the endotoxic activity per IL-8 secretions (approximately 100e400 pg/106 cells) at a 1000 EU/ dry weight of E. coli LPS as shown in Fig. 2. Acinetobacter spp. mL dose. The last group including two Pseudomonas sp., including A. lwoffii are known not just as opportunistic pathogens but also ubiquitous habitant in biofilm in water distribution systems. (Scott and Pepper, 2010; Vaz-Moreira et al., 2013; Kelly et al., 2014). This result suggests that inflammatory potency can increase with the regrowth of Acinetobacter sp. in distribution systems even though the endotoxin activity remains low.

3.4. Comparison of inﬂammatory potencies of river water and BAC efﬂuent

The IL-8 and TNF-a secretions from THP-1 stimulated with the LPSs purified from the river water and the BAC effluent samples are also presented in Figs. 3 and 4, respectively. Recently data on endotoxin levels in various aquatic environments has been collected all over the world. For example, several studies reported that endotoxin levels in drinking water derived from treated surface water were in the range of 1e10 EU/mL (Ohkouchi et al., 2009; Can et al., 2013). Usually the endotoxin levels in surface water were Fig. 2. Comparison of endotoxin contents based on dry weight in purified LPS samples a few hundred EU/mL, but they rose above 1000 EU/mL if the sur- from pure-cultured bacteria/cyanobacteria and water samples. face water was impacted by effluents from sewage treatment plants 28 Y. Ohkouchi et al. / Toxicon 97 (2015) 23e31

Fig. 3. The effects of purified LPSs from various pure-cultured bacteria/cyanobacteria and water samples on the IL-8 secretion of the THP-1 cell-line. The LPSs purified from (a) M. fujisawaense ( ), (b) A. lwoffii (C), M. aeruginosa (-), A. delafieldii (B), river water (A), BAC effluent (◊), and (c) E. coli (▫), Synechococcus sp. (-), P. fluorescens (:), P. aeruginosa (▵), A. commune (C) were used for 6 h stimulation. The cytokine data represent means of two independent assays.

(Ohkouchi et al., 2007) or cyanobacterial bloom (Rapala et al., prove that Acinetobacter lwoffii and indigenous bacteria in aquatic 2002). Based on our result, relatively strong cytokine secretions environments have relatively high cellular endotoxin contents and caused by stimulation with river water and BAC effluent were reveal a common pattern by which significant cytokine secretions observed at normal endotoxin levels in an aquatic environment, can be induced in human cells. around 100 EU/mL (104e105 cells/mL as total cells). Of special note Thus, LPSs purified from both the river water and the BAC is that the BAC effluent LPS induced a moderate inflammatory effluent induced much stronger inflammatory responses at doses of response (approx. 250 pg/106 THP-1 cells) even at 10 EU/mL, which 10e5000 EU/mL than did the LPSs of most lab-cultured bacteria/ is the same endotoxin level as in drinking water. At LPS doses above cyanobacteria. At the same time, both water samples induced very 500 EU/mL, cytokine secretions were increased in a dose- high TNF-a secretions as well as IL-8 secretions like Acinetobacter dependent manner by stimulation with the river water LPS, LPS rather than the LPSs purified from other lab-cultured bacteria/ whereas cytokine levels did not increase significantly by stimula- cyanobacteria. The reasons for such stronger responses by stimu- tion with the BAC effluent LPS. lation with the indigenous bacterial LPSs have not yet been iden- Principal component analysis (PAC) was then applied to group tified, but there are several possibilities including the following: the purified LPS samples, except Methylobacterium fujisawaense, 1) In aquatic environments, bacteria like Acinetobacter lwoffii, based on their cytokine profiles at a dose of 1000 EU/mL and their which have LPSs that cause strong inflammatory responses in hu- cellular endotoxin contents. The endotoxin dose of 1000 EU/mL man monocytes, are present in abundance; 2) low water temper- was chosen by considering actual endotoxin levels in aquatic en- ature or low nutritional/osmotic conditions in aquatic vironments. The results of the analysis are presented in Fig. 5. The environments can increase the potency of indigenous bacterial PCA score plot showed that the river water LPS, the BAC effluent LPSs; and 3) some substances that enhance inflammatory re- LPS, and Acinetobacter lwoffii LPS formed a group distinct from the sponses exist in those purified samples. The third possibility will be other bacterial/cyanobacterial LPSs. This group represented bacte- discussed in a later section. Kawahara et al. (2002) and Dentovskaya ria that induced strong cytokine secretions in human monocytes et al. (2008) reported that the changes in the structure and in- although their endotoxic activities per cell were moderate (approx. flammatory potency of Yersinia pestis LPS depend on the growth 10 3 EU/cell). In contrast, Aquabacterium commune LPS exhibited temperature. According to their results, the purified LPS from very high endotoxin activity but very low induction of inflamma- Y. pestis grown at 25 or 27 C could induce stronger TNF-a secretion tory responses and was distant from the other LPSs. These results in mouse and human macrophages than that grown at 37 C. They

Fig. 4. The effects of purified LPSs from various pure-cultured bacteria/cyanobacteria and water samples on the TNF-a secretion of the THP-1 cell-line. The LPSs purified from (a) M. fujisawaense ( ), (b) A. lwoffii (C), M. aeruginosa (-), A. delafieldii (B), river water (A), BAC effluent (◊), and (c) E. coli (▫), Synechococcus sp. (-), P. fluorescens (:), P. aeruginosa (▵), A. commune (C) were used for 6 h stimulation. The cytokine data represent means of two independent assays. Y. Ohkouchi et al. / Toxicon 97 (2015) 23e31 29

caused by water temperature or other environmental conditions in the aquatic environment, LPS structures of indigenous bacteria may shift toward the more potent.

3.5. Effect of contaminants in puriﬁed LPSs on TNF-a secretions

As discussed above, there is a possibility that contaminated substances that remained in the purified LPSs of the river water and the BAC effluent contribute to the elevation of TNF-a secretions. In order to exclude this possibility, the cytokine secretions from THP-1 stimulated with each LPS with/without polymixin B as an inhibitor of endotoxins were evaluated. The results are presented in Fig. 6. Except for the LPS samples from the BAC effluent, the addition of polymixin B significantly inhibited both cytokine secretions. This result proved that the LPS components in the purified sample from river water greatly contributed to the cytokine elevations, including TNF-a, as with the Acinetobacter LPS. In contrast, the cytokine levels stimulated with the BAC effluent LPS were not affected by the addition of polymixin B. The contribution of contaminant substances such as peptidoglycan or lipoteichoic acid to the strong cytokine secretions was suspected. However, the contribution of fi Fig. 5. Principal component analysis of various LPS samples puri ed in this investi- contaminant substances in the BAC effluent LPS was unlikely gation. The first and second principal components explained 63.8% and 33.4% of the total variability, respectively. because several researches reported that more than 100-fold amounts of contaminated substances such as peptidoglycan or lipoteichoic acid were required to induce the same level of cytokine also proved that hexaacylated lipid-A, which is synthesized in secretions with LPSs. Another reason that polymixin B failed to bacteria only grown at low temperatures, contributes to the inhibit the secretions of both types of cytokines, IL-8 and TNF-a,in potentiation of inflammatory responses. In addition, Ernst et al. that sample may be the lower affinity of polymixin B with the LPSs (2006) indicated that the LPS structure of Pseudomonas aerugi- in the BAC effluent. Kasuga et al. (2011) reported that different nosa change depending on temperature for cultivation. Especially, bacterial communities from those in the water sources were hexa-acylated LPS was dominant at 15 C while after growth at developed on the BAC surface installed after the ozonation process. elevated temperature penta-acylated LPS was dominant. They Such a difference could have resulted in the diversity in the LPS suggest that the dominance of hexa-avylated LPS at lower tem- chemical structures in the BAC effluent sample. At the same time, perature could be attributed to lower deacylase activity, which is several researchers also suggested that the surrounding conditions deficient in one-third of clinical isolates. In our investigation, both such as the water temperature or osmosis can also contribute to the the water samples from the river and from the BAC process were diversity of the chemical structures of LPSs in the environmental taken in winter when the water temperature was below 10 C, samples (Merino et al., 1998; Suomalainen et al., 2010). On the whereas all bacteria/cyanobacteria except for Escherichia coli were other hand, Coyne and Fenwick (1993) reported that the ability of cultured at 20 C in the laboratory in order to make conditions polymixin B to inhibit TNF-a synthesis in macrophages had closer to the natural aquatic environment. Based on the above in- considerable variability. For example, they showed that polymixin formation, as a consequence of a decrease in deacylase activity B of 1 IU/mL completely inhibited TNF-a synthesis in macrophages in the presence of LPS from Escherichia coli B4:O111. The relative

Fig. 6. Effects of polymixin B addition as an inhibitor of endotoxin on cytokine secretion in the THP-1 cell line. IL-8 and TNF-a were determined after 6 h stimulation with LPS samples and with LPS samples pretreated with polymixin B, respectively. The cytokine data represent means with standard deviations of four independent assays. Asterisks indicate a signiﬁcant difference in comparison to the samples without the polymixin B pretreatment. 30 Y. Ohkouchi et al. / Toxicon 97 (2015) 23e31

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