| Tris(2-chloroethyl)phosphate(TCEP),a halogenated organophosphate flame retardants and plasticizer,has been widely used in a variety of commercial products,including plastic,textiles,electric products,construction and decoration materials.It is easily emitted to the environment owing to it is not covalently bound to the high polymer materials.TCEP as a micropollutant has been detected in drinking water in addition to air,precipitation,surface water and groundwater.However,limited data are available on seasonal variations of TCEP in water organic extracts.Experiments showed that TCEP(?175mg/kg bw/d)promoted hepatocarcinogenesis in Scl:dd Y mice.TCEP(?3.12mg/L)caused growth inhibition,cycle arrest and senescence of human liver cell lines(such as human embryonic liver cells(L02)and human hepatoma cells(Hep G2)).Polycyclic Aromatic Hydrocarbons(PAHs)are emitted during incomplete combustions of wood fires,gaseous fuels and tobacco.They widely exists in the environment due to its stable chemistry properties.Benzo[?]pyrene(Ba P)is metabolized by several cytochrome P450(CYP)enzymes to become the ultimate carcinogen along with induction of oxidative stress and inflammatory response in human body.Ba P is accepted as the most powerful carcinogen and mutagen,and classified as a group 1 carcinogen by International Agency for Research on Cancer.Human beings are simultaneously exposed to TCEP and Ba P through inhalation,oral intake and dermal absorption in daily life)and may occur toxic adverse events.However,the mechanisms underlying effect of TCEP combined with Ba P still need to be illustrated.Inflammatory response is accepted as an essential event in response to environmental pollutants.Accumulated data reveal that activated Ba P can induce the activations of inflammatory cytokines,such as interleukin(IL)-1,IL-6 and IL-8,thus leading to occurrences of and developments of tumors.An animal experiment showed that TCEP induced incidence of altered eosinophilic foci in the livers of male B6C3F1 mice treated with TCEP(175 and 350mg/kg bw/day)for two years.However.the mechanisms underlying inflammatory effects of TCEP combined with Ba P remains unclear.Based on the scientific clues regarding the toxic effects of TCEP alone on the L02 and Hep G2,the present study aimed to investigate seasonal variations of TCEP concentrations in water samples from the two drinking water plants in Wuhan city,China,cellular toxicities of water organic extracts and molecular mechanisms for toxic effects of TCEP combined with Ba P and in Hep G2 cells.The study included two parts.Part One: TCEP concentrations in water organic extracts and cytotoxicities of water organic extractsObjectives: We investigated seasonal variations of TCEP concentrations in the raw,finished and tap water samples from two drinking water treatment plants(DWTPs),and evaluated the cytotoxicities and apoptosis/necrosis induced by water organic extracts(OEs).Methods: The raw,finished and tap water samples(n=40)were collected from DWTP A(Yangtze river served as raw water)and DWTP B(Hanjiang river served as raw water)in the spring,summer,autumn and winter seasons.OEs in water samples were enriched by solid phase extraction.TCEP concentrations in the OEs were measured by gas chromatography coupled with mass spectrometry(GC/MS).We treated L02 cells with different concentrations of OEs(12.5,25.0,50.0 and 100.0m L/m L cell culture)or dimethyl sulfoxide(DMSO,as solvent control,final concentration(V/V): 0.1%).At 24 and 48 h after treatment,we assessed cell viabilities using the 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay,and evaluated apoptosis and necrosis rates in the OEs-treated L02 cells by flow cytometry.Results: The results showed that seasonal TCEP concentrations in the raw,finished and tap water samples from DWTP A ranged from below the MDL or MDL to 676ng/L,33ng/L to 78ng/L and 35ng/L to 257ng/L,respectively.Seasonal TCEP concentrations in the raw,finished or tap water samples from DWTP B ranged from below the MDL or MDL to 505ng/L,below the MDL or MDL to 104ng/L and below the MDL or MDL to 130ng/L,respectively.The maximal TCEP concentrations in the raw water samples(676ng/L for DWTP A and 505ng/L for DWTP B)appeared in the winter.The maximal TCEP concentrations in tap water samples appeared in the autumn for DWTP A(257ng/L)and in the spring for DWTP B(130ng/L),respectively.OEs in raw water samples from the Yangtze river in the summer and from Hanjiang river in the winter showed the strongest cytotoxicities.OEs from the finished and tap water samples in the summer and autumn from these two DWTPs exhibited stronger cytotoxicities than those in the spring and winter.Conclusions: TCEP was not sufficiently removed during the process of drinking water treatment systems in these two DWTPs.The TCEP concentrations in the raw water samples from DWTPs A and B in the winter and spring were higher than those in the summer and autumn.OEs(100m L/m L cell culture)in the raw water samples in four seasons from DWTPs A and B induced apoptosis of L02 cells.Part Two: Effects of TCEP combined with Ba P on the releases of IL-6 and IL-8 from Hep G2 cells via the EGFR-ERK1/2 signaling pathwayObjectives: The mechanisms underlying effects of TCEP combined with Ba P on the releases of IL-6 and IL-8 in Hep G2 cells via the activation of EGFR/MAPK pathway were investigated.Methods: Human hepatoma Hep G2 cells were treated with either TCEP(3.12,12.50,50.00 or 200.00mg/L)alone,Ba P(50?M)alone or TCEP plus Ba P for 24 and 48 h.DMSO(solvent control,final concentration(V/V): 0.1%)served as the solvent control.We measured the viabilities of the treated cells using MTT assay,and oxidative stress indicators in the treated cells,including malondialdehyde(MDA),glutathione peroxidase(GSH-Px),oxygen radical absorbance capacity(ORAC),hydroxygen radical absorbance capacity(HORAC)using the corresponding detection assay kit.Additionally,we detected apoptosis rates of the treated cells by flow cytometry;the secretions of IL-6 and IL-8 proteins in the culture medium were measured by the enzyme-linked immunosorbent assay;the transcriptional levels of IL-6 and IL-8 genes were determined by real-time polymerase chain reaction,and expression of proteins(including EGFR,p-EGFR,ERK1/2,p-ERK1/2,p38,p-p38,JNK,p-JNK,STAT3,p-STAT3,p65,p-p65)involved in inflammation-associated signaling pathway were measured by western blotting.Thereafter,at 1h after treatment with an inhibitor of AG1478(a EGFR inhibitor),U0126(a ERK inhibitor)or SB203580(a p38 inhibitor),Hep G2 cells were treated with 50?M Ba P alone,50mg/L TCEP alone or each of the inhibitors plus 50mg/L TCEP and 50?M Ba P,the secretions of IL-6 and IL-8 proteins in the culture medium and the transcriptional levels of IL-6 and IL-8 genes were measured again.Results: At 24 and 48 h after Hep G2 cells were treated with TCEP at different concentrations(3.12,12.50,50.00 or 200.00mg/L)alone,Ba P(50?M)alone or TCEP plus Ba P,compared to the solvent control,decreased cell viabilities were found in the groups of 200mg/L TCEP alone and co-treated groups with ?3.12mg/L TCEP plus 50?M Ba P(P<0.05),however,the expression of IL-6 and IL-8 at the transcriptional and protein levels were upregulated(P<0.05).Moreover,expression of p-EGFR protein was upregulated in the co-treated cells with TCEP at different concentrations(3.12,12.50 or 50.00mg/L)plus 50?M Ba P(P<0.05),whereas,expression of p-ERK and p-p38 proteins were upregulated in the co-treatment of ?3.12mg/L TCEP plus 50?M Ba P(P<0.05).Compared with the corresponding cells treated with TCEP alone,decreased viabilities were found in the co-treated cells with ?3.12mg/L TCEP plus 50?M Ba P(P<0.05),and expression of IL-6 and IL-8 at transcriptional and protein levels were upregulated(P<0.05).Expression of p-EGFR protein in the co-treated cells with TCEP at different concentrations(3.12,12.50 or 50.00mg/L)plus 50?M Ba P were upregulated(P<0.05),however,expression of p-ERK and p-p38 proteins were upregulated in the co-treated cells with ?3.12mg/L TCEP plus 50?M Ba P(P<0.05).Compared with the co-treated cells with 50mg/L TCEP plus 50?M Ba P,inhibitors(AG1478,U0126 and SB203580)down-regulated expression of IL-6 and IL-8 at the transcriptional and protein levels(P<0.05),however,AG1478 down-regulated expression of p-ERK protein was only observed in the co-treated cells with plus 50mg/L TCEP plus 50?M Ba P.Conclusions: The stronger inhibitory effects on the viabilities of co-treated Hep G2 cells with TCEP plus Ba P were observed rather than that of cells with ?3.12mg/L TCEP alone or 50?M Ba P alone.The secretions of IL-6 and IL-8 proteins were triggered in the co-treated Hep G2 cells with TCEP(3.12,12.50,50.00 or 200mg/L)plus 50?M Ba P through the EGFR-ERK1/2 signaling pathway. |
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