immunotoxicity of pyrethroid metabolites in an in vitro model
TRANSCRIPT
tal Toxicology
Environmental Toxicology and Chemistry, Vol. 29, No. 11, pp. 2505–2510, 2010# 2010 SETAC
Printed in the USADOI: 10.1002/etc.298
Environmen
IMMUNOTOXICITY OF PYRETHROID METABOLITES IN AN IN VITRO MODEL
YING ZHANG,y MEIRONG ZHAO,z MEIQING JIN,y CHAO XU,z CUI WANG,z and WEIPING LIU*yzyMOE Key Lab of Environmental Remediation and Ecosystem Health, Zhejiang University, Hangzhou 310027, People’s Republic of China
zResearch Center of Environmental Science, Zhejiang University of Technology, Hangzhou 310032, People’s Republic of China
(Submitted 10 December 2009; Returned for Revision 20 March 2010; Accepted 16 May 2010)
* T(wliu@
Pub(wileyo
Abstract—Risk assessment of man-made chemicals such as pesticides are mainly focused on parent compounds, and relatively little isknown about their metabolites, especially with regard to target organ damages such as immunotoxicity. In the present study, theimmunotoxicity of five synthetic pyrethroids (SPs) and three common metabolites was evaluated using an in vitro model by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cytoflow, and enzyme-linked immunosorbent assay (ELISA).Cell viability and apoptosis assays showed that both SPs and their metabolites possessed cytotoxicity to the monocytic cells. Thealdehyde and acid derivatives were more effective than the other compounds at cytotoxicity, with inhibition of cell viability by 56.8 and50.6% at 10�5 mol L�1, and induction of 8.52 and 8.81% cell apoptosis, respectively. Exposure to SPs and their metabolites also led tochanges in the secretion levels of tumor necrosis factor a (TNF a) and interleukins (ILs), and again the metabolites showed strongereffects than the parent compounds. The aldehyde derivative upregulated IL-12p70 level by 1.87-fold, and the alcohol and acid derivativeincreased the secretion of TNF a 5.88 and 7.96-fold, relative to the control group. In the in vitro model, the common metabolites of SPsclearly exerted greater immunotoxic effects to monocytes than the intact parent compounds. Results from the present study suggested theneed for considering metabolites in achieving more comprehensive health risk assessment of man-made chemicals, including targetorgan toxicities such as immunotoxicity. Environ. Toxicol. Chem. 2010;29:2505–2510. # 2010 SETAC
Keywords—Cytokine Cytotoxicity Metabolites Monocytes Pyrethroids
INTRODUCTION
Once released into the environment, organic compoundssuch as pesticides are subject to chemical or biochemicaltransformations, leading to the formation of metabolites. Forpesticides, both parent compounds and their metabolites mayexert toxic effects on humans and other mammals. In someinstances, the transformation products of pesticides are moreprevalent in the environment or have higher toxicities thanthe parent compound [1,2]. However, in general, most riskassessment practices focus only on parent compounds, andrelatively few cases consider pesticide metabolites [3]. Forexample, at present, essentially no knowledge is available onthe immunotoxicity of pesticide metabolites.
The immune system is in a complex balance interacting withother systems and plays a critical role in maintaining the healthof humans and animals. It consists of a complicated network ofcells and mediators, such as cytokines, to act on innate andinducible immune functions in a highly regulated manner. Bothsuppression and enhancement of immune functions by certainchemicals is thought to exhibit potential immunotoxicity of thechemicals. The immune system appears to be a sensitive andcomplex target for pesticides [4]. In view of their widespreaduse and distribution, exposure to pesticides may represent animportant cause for immune system disruptions and may resultin induced immunomodulations that endanger humans andanimals [5].
Synthetic pyrethroids (SPs), recognized as two differenttypes by the absence (type I) or presence (type II) of an a-
o whom correspondence may be addressedzjut.edu.cn).
lished online 9 July 2010 in Wiley Online Librarynlinelibrary.com).
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cyano group, are among the most commonly used insecticides[6]. The popularity of SPs is attributed to their high efficacy toinsects, low environmental mobility, and relatively low mam-malian and avian toxicity [7]. However, an increasing numberof studies show that SPs are capable of disrupting hormonalactivities [8], causing oxidative stress [9], inducing immunesuppression [10], and inhibiting signal transduction [11]. Pyr-ethroids are metabolized oxidatively and hydrolytically to forma number of primary and secondary metabolites [12]. Threeintermediates, i.e., 3-phenoxybenzoic alcohol (PBCOH), 3-phenoxybenzaldehyde (PBCHO), and 3-phenoxybenzoic acid(PBCOOH), are common to many SPs. These metabolites havebeen found in animal and human tissues, blood, and urine[13,14], as well as in the environment as microbial transfor-mation products [15]. For example, a study showed thatPBCOOH was the most frequently detected metabolite in82% of the urine samples collected from children [16]. Previousstudies showed that some SPs displayed immunotoxicologicaleffects [17] and endocrine disrupting activities [8], which, ifcoupled with the recent finding that metabolites of SPs possessendocrine disrupting activities [18,19], points to a likelihood forSP metabolites to induce immunotoxicity like SPs. In addition,because SP metabolites are much more polar than the parentcompounds, they may be more easily absorbed and thereforecontribute to increased immunotoxicity to animals and humans.
The primary objective of the present study was to evaluatethe immunotoxicity of SPs and their common metabolites. Awell-known human monocytic cell line U937 [20] was usedas the in vitro model for the assays. The monocytes play asignificant role in the innate immune system, which secretescytokines such as tumor necrosis factor m (TNF a) and inter-leukins (ILs) to take part in immune reaction. It is expectedthat both the results and approaches developed in thepresent study may be useful for better understanding the
2506 Environ. Toxicol. Chem. 29, 2010 Y. Zhang et al.
immunotoxicity of SPs and their metabolites, and other toxiceffects in general.
MATERIALS AND METHODS
Chemicals and reagents
Permethrin (PM, >95%), d-phenothrin (d-PN, >94.9%),PBCOH (>98%), PBCHO (>97%), and PBCOOH (>98%)were purchased from Sigma Chemical. Cypermethrin (CP,>95%) and d-cyphenothrin (d-CPN, >92%) were obtainedfrom Xinhuo Technical Institute. Lambda-cyhalothrin (LCT,>98%) was purchased from Danyang Agrochemicals. Struc-tures of all these compounds are given in Figure 1. All testedcompounds were dissolved in HPLC grade ethanol (Tedia) andkept at 4 8C in the dark as stock solutions. Other chemicals orsolvents used in the present study were of cell culture, HPLC, oranalytical grade.
Cell culture and treatments
The U937 cells, obtained from the State Key Laboratory ofPharmaceutical Biotechnology, Nanjing University, were cul-tured in RPMI-1640 medium (HyClone) supplemented with10% of fetal bovine serum (FBS, HyClone) at 37 8C in ahumidified CO2 incubator (Thermo Electron) of 5% CO2 and95% air. The culture medium was refreshed every 3 d, andreplaced with the experimental medium (RPMI-1640 contain-ing 2% FBS) for 1 d to reduce the effect of serum beforetreatment. The cells were then treated with the dosing medium(the experimental medium along with test compound at con-centrations of 10�9–10�5 mol L�1) for 3 d (for cell viabilityassay) or 2 d (apoptosis and cytokine analysis). A series of testsolutions were prepared in ethanol, with the final solventconcentrations less than 0.1% by volume. Ethanol (0.1% v/v)was used as the negative control.
Assessment of cell viability
The cell viability was determined by MTT assay based uponthe reduction of thiazolyl blue (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Amresco). Cells in an
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The Metabolites
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Fig. 1. Chemical structures of synthetic pyrethroids (SPs) and theirmetabolites.
exponential growth status were seeded in 96-well plates at adensity of 5� 104 cells mL�1 for pretreatment of 24 h, and thenthe medium was changed to the dosing medium containing testsolutions at a range of concentrations. After 3-d exposure, MTTsolution (5 mg mL�1 phosphate-buffered saline [PBS]) wasadded into wells, followed by incubation at 37 8C for 4 h.The medium was then removed from the wells, and 150 mlDMSO was added per well. After mixing on a micro-mixer for10 min, the absorbance was measured at a wavelength of570 nm with background subtraction at 650 nm using a Bio-Rad Model 680 microplate reader (Bio-Rad Laboratories). Thetreatments were all repeated at least three times. The resultswere expressed in the relative viability, which was the ratio ofeach exposure group over the vehicle control.
Analysis of cell apoptosis
In the early stage of apoptosis, changes occur at the cellsurface such as translocation of phosphatidylserine [21], whichcan be analyzed by using Annexin-V-Fluorescein and ProidiumIodide (PI). The SPs and their metabolite-induced cell apoptosiswere determined by the Annexin-V-FLUOS staining kit (Roche)according to the manufacturer’s protocol. High Annexin-V-Fluorescein and low PI staining indicates early apoptotic cells,whereas high PI staining indicates necrotic cells. Cells at aninitial concentration of 5� 104 per well were incubated withthe vehicle control or test solutions at the concentrationof 10�6 mol L�1 in 6-well plates for 48 h. After harvest andwashing twice with cold PBS, cells were resuspended in 100 mlAnnexin-V-FLUOS labeling solution (containing 2 ml Annexin-V-FLUOS labeling reagent and 2 ml PI) and incubated for 15 minat room temperature in the dark. The final samples wereanalyzed on a flow cytometer (Becton Dickinson).
Measurement of cytokine secretion
Assessment of cytokine, the molecules in response to reg-ulating various processes including immunity, inflammation,apoptosis, and hematopoiesis, is a valuable tool for evaluatingchemical exposure effects on the immune system [22]. Tofurther investigate the molecular mechanisms of toxicity, theeffects of SPs and their metabolites on cytokine secretionwere measured. Cells were cultured in 24-well plates withthe vehicle control or 10�6 mol L�1 test solutions for 48 h.Cell culture supernatants were collected and stored at �20 8C.The proinflammatory cytokines TNF a and IL-6, immunore-gulatory cytokine IL-10, and also immune response regulatorIL-12p70 were measured by commercial enzyme-linked immu-nosorbent assay (ELISA) kits (Cusabio) according to the man-ufacturer’s instructions. Each measurement was repeated atleast four times.
Statistical analysis
The results were presented as mean�SD and tested forstatistical significance by analysis of variance (ANOVA) usingSPSS 16.0. Differences were considered statistically significantwhen p value was less than 0.05 or 0.01.
RESULTS
Inhibitory responses in cell viability
The MTT assay for cell vitality was first carried out toinvestigate the responses of U937 cell line to SPs and theirmetabolites. From the dose–response relationships, the testcompounds displayed an inhibitory effect on U937 viabilityin a concentration-dependent manner (Fig. 2). The viabilities of
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Fig. 2. The effects of synthetic pyrethroids (SPs) and their metabolites on the viability of U937 cell lines. The U937 cells were exposed to a series of concentrationsof Permethrin (PM) (A), d-phenothrin (d-PN) (B), Cypermethrin (CP) (C), Lambda-cyhalothrin (LCT) (D), d-cyphenothrin (d-CPN) (E), 3-phenoxybenzoicalcohol (PBCOH) (F), 3-phenoxybenzaldehyde (PBCHO) (G), and 3-phenoxybenzoic acid (PBCOOH) (H) for 72 h followed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are presented as mean�SD of at least three independent assays (� indicates p< 0.05, and �� indicatesp< 0.01, compared to negative control by analysis of variance [ANOVA]).
Fig. 3. Evaluation of apoptotic cells by the Annexin-V staining assay. TheU937 cells incubated with vehicle control (A) or 10�6 mol L�1 PM (B), d-PN(C), CP (D), LCT (E), d-CPN (F), PBCOH (G), PBCHO (H), and PBCOOH(I) for 48 h were stained by Annexin-V and PI and then analyzed by flowcytometer. The x axis represents increasing Annexin-V fluorescence(relative light unit), and the y axis represents increasing Proidium Iodide(PI) fluorescence (relative light unit). The subpopulations in quadrants II–IVrepresent necrotic cells (II), living cells (III), and early apoptotic cells (IV).See Figure 2 for acronym key.
Immunotoxicity of pyrethroid metabolites Environ. Toxicol. Chem. 29, 2010 2507
U937 were significantly inhibited at 10�6 or 10�5 mol L�1 forall the compounds. Among the five different SPs, CP (type II)was more toxic than the other SPs at 10�6 mol L�1 (p< 0.05).At 10�5 mol L�1, PM, d-PN, CP, LCT, and d-CPN decreasedcell viability to 90.0, 73.2, 67.0, 76.6, and 65.3%, and thedifference between PM and the other four SPs was statisticallysignificant (p< 0.05). The three metabolites also caused inhib-ition of cell growth within the range of 10�8–10�5 mol L�1.Moreover, PBCHO and PBCOOH were much more toxic thanthe parent compounds and PBCOH (p< 0.001 for PBCHO andp< 0.05 for PBCOOH) at 10�5 mol L�1, with cell growthinhibited by 56.8 and 50.6%, respectively. The results showedthat the metabolites induced suppression of cell viability stron-ger than the parent compounds.
Induction of U937 cells apoptosis
As the inhibitory responses in cell viability may be attributedto arrest of cell cycles and induction of apoptosis, the Annexin-V- FLUOS staining kit was used to determine the effects of SPsand their metabolites at 10�6 mol L�1 on U937 cell apoptosis.The results showed that the numbers of early apoptotic cellsin the bottom right quadrant increased after exposure to SPs,suggesting that SPs were able to induce visible early apoptosisof U937 cells (Fig. 3). No significant difference existed betweenthe test groups and the negative control in the percentage ofnecrotic cells. The percentages of cells stained as Annexin-V� /PI� (living cells), Annexin-Vþ /PI� (early apoptoticcells), and Annexin-Vþ /PIþ (necrotic cells) are presentedin Table 1. On average, PM, d-PN, CP, LCT, and d-CPNtreatments resulted in 4.88, 7.49, 9.50, 8.51, and 7.53% apop-
Table 1. Percentage of apoptotic cells induced by synthetic pyrethroidsand their metabolites using Annexin-V staining assaya
Compound Quadrant I Quadrant IIb Quadrant IIIc Quadrant IVd
Control 0.09 1.14 95 3.75PM 0.03 1.2 93.9 4.88d-PN 0.15 1.6 90.8 7.49CP 0.01 1.51 89 9.5LCT 0 1.97 89.5 8.51d-CPN 0.04 1.33 91.1 7.53PBCOH 0.04 1.02 91 7.98PBCHO 0.03 1.47 90 8.52PBCOOH 0 0.94 90.3 8.81
a See Figure 2 for acronym key; PI¼ proidium iodide.b The percentage of necrotic cells with low Annexin-V and high PI staining.c The percentage of living cells with low Annexin-V and low PI staining.d The percentage of early apoptotic cells with high Annexin-V and low PI
staining.
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Fig. 4. Assessment of cytokine secretions of cell culture supernatants. TheU937 cells were cultured with vehicle control or 10�6 mol L�1 test solutionsfor 48 h, and secretions of interleukin-6 (IL-6), interleukin-10 (IL-10) (A),interleukin-12p70 (IL-12p70) (B), and tumor necrosis factor a (TNF a) (C) inculture supernatants were measured by enzyme-linked immunosorbent assay(ELISA). � indicates p< 0.05, and �� indicates p< 0.01, relative to eachsolvent control. Different lowercase letters indicate a significant difference(p< 0.05) between the parent compounds and their metabolites (a forPBCOH; b for PBCHO; c for PBCOOH). Different capital letters above errorbars indicate a significant difference (p <.05) between three metabolites,while the same letter indicates no significant difference. See Figure 2 foracronym key.
2508 Environ. Toxicol. Chem. 29, 2010 Y. Zhang et al.
totic cells, respectively, while the negative control group hadonly 3.75% apoptotic cells. Type II SPs appeared to haveinduced more early apoptosis than type I SPs. The metabolitesdisplayed similar effects of inducing cell apoptosis, andPBCOH, PBCHO, and PBCOOH were found to induce 7.98,8.52, and 8.81% early apoptotic cells, respectively. This obser-vation suggested that the common metabolites were capable ofcausing the same or more intensive apoptosis than their parentcompounds in U937 cells.
Alteration of cytokine secretion
Synthetic pyrethroids and their metabolites may not onlyinduce cytotoxicity, but also alter immune functions through thealteration of cytokines such as TNF a and ILs that participate incomplex interactions with cell viability in immune cells. Thelevels of IL-6, IL-10, IL-12p70, and TNF a in U937 monocyteswere determined using ELISA kits. With respect to ILs, only theIL-12p70 level was altered by SPs and their metabolites. Asshown in Figure 4A, the secretion of IL-10 decreased slightly inthe test groups of CP, d-CPN, PBCOH, and PBCOOH, but thedifferences were not significant. Moreover, no significant dif-ferences were observed in the concentrations of IL-6 among thedifferent treatments (data not shown). In addition, the levels ofIL-12p70 were not significantly affected after PM, CP, and LCTtreatments, but were increased after exposure to d-PN and d-CPN ( p< 0.05) (Fig. 4B). After the treatment of PBCHO, IL-12p70 level was upregulated to 1.87-fold of the negativecontrol, which was significantly higher (p< 0.05) than theparent compounds. PBCOOH also increased the level of IL-12p70 ( p< 0.01), although the increase was not statisticallysignificant when compared to the parent compounds.
Exposure of U937 cells to SPs and their metabolites gen-erally resulted in an increased secretion of TNF a, except ford-CPN (p< 0.05) (Fig. 4C). Permethrin, LCT, and PBCOHinduced similar levels of TNF a production, ranging from 4.95-to 5.88-fold increases relative to the negative control. Theseincreases were also higher than PBCHO (p< 0.05) (1.98-foldincrease relative to the negative control). Furthermore,PBCOOH induced the highest increment of TNF a secretion,equaling 7.96-fold relative to the control group. The increaseinduced by PBCOOH was significantly higher than all theparent compounds ( p< 0.05). Overall, the results from cytokineanalysis showed that the common metabolites were capable ofinducing similar or more intensive effects on cytokine secretionthan the parent compounds. These results also implied that the
Immunotoxicity of pyrethroid metabolites Environ. Toxicol. Chem. 29, 2010 2509
levels of IL-12p70 and TNF a in monocytes may be sensitiveendpoints for the evaluation of immunotoxicity of SPs and theirmetabolites.
DISCUSSION
With the widespread use of pesticides, more comprehensiverisk assessment by considering their environmental metabolitesis imperative. Although limited studies previously showedimmunotoxicity of SPs using in vivo and in vitro models, thepresent study demonstrated for the first time, to our knowledge,that the common metabolites of SPs were capable of inducingsimilar or more intensive immunotoxic effects than the parentcompounds.
In mammals, SPs are rapidly metabolized to less lipophilicand more readily excreted metabolites [19]. For instance, theelimination was nearly complete within 5 d of exposure for mostSPs following inhalation exposure, while the majority of thedose was eliminated in the first 1 to 2 d following oral exposureof humans or animals ([23]; http://www.atsdr.cdc.gov/toxpro-files/tp155.html). Urine analysis showed no presence of SPs,however, the metabolites were detected within several hoursafter exposure depending on chemical structures. Studiesshow that SPs can be metabolized in liver microsomes, hepaticcytosol, serum, and small intestinal microsomes [24,25]. Themost important metabolism of most SPs occurring in livermicrosomes is cleavage of the central ester linkage, whichproduces a cyclopropane acid and an alcohol moiety (Fig. 5).The alcohol moiety is then hydroxylated to produce PBCOHthat is further oxidized to PBCOOH using PBCHO [26]. Sub-sequently, these metabolites undergo conjugation processes toproduce glucoronides of the carboxylic acid or sulfates of thephenols, which are excreted in the urine. In the natural environ-ment and higher plants, SPs are also metabolized or degraded toform these common metabolites ([27]; http://ace.ace.orst.edu/info/extoxnet/pips/pyrethri.htm).
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Fig. 5. Metabolism of synthetic pyrethroids (SPs) in mammals. PBCOH¼3-phenoxybenzoic alcohol; PBCHO¼ 3-phenoxybenzaldehyde; PBCOOH¼3-phenoxybenzoic acid.
Evaluation of cell growth and apoptosis on the target cellsafter exposure suggested that most of the parent compoundsinhibited cell viability and induced monocyte apoptosis, imply-ing that SPs possessed cytotoxicity to the monocytic cells. Thisfinding was in agreement with some previous in vitro and invivo studies on SPs [28,29], as well as studies showing thatpermethrin and deltamethrin increased apoptotic or necroticcell death in thymocytes [29,30]. Among the three metabolites,PBCHO and PBCOOH significantly inhibited the U937 cellgrowth within the concentrations of 10�8 to 10�5 mol L�1,showing that the metabolites possessed much higher toxicitythan the parent compounds. The metabolites further displayedsimilar or more intensive apoptosis than the parent SPs. Bothobservations clearly suggested that the SP metabolites werecapable of causing higher cytotoxicity than the parent SPs inmonocytes.
Assay of cytotoxicity alone may not be adequate to showpesticide-induced immunotoxicity, because cytokines also playa paramount role in mediating cell–cell communication ofinflammatory and immune responses. Measurement of immuneresponses of cytokine secretions is, therefore, an importantaspect in defining pesticide immunotoxicity. Analysis of theeffects of SPs and their metabolites on cytokine stimulationshowed that exposure to PBCOH and PBCOOH resulted ingreater disruption of cytokines of monocytes than the othercompounds. Although no obvious effect was noted on thesecretion of IL-10 and IL-6, specific metabolites induced moreintensive effects on the secretion of TNF a and IL-12p70 thanthe parent compounds. These results suggested that the commonSP metabolites were capable of altering immune functions inaddition to inducing cytotoxicity in human monocytes. Thecurrent understanding of interactions between cytokines is stillnot clear, and therefore more research is needed to furtherinvestigate the underlying mechanisms for these effects.
Monocytes are known to protect the body from a series ofpathogens and xenobiotics by releasing cytotoxic and proin-fiammatory substances (e.g., TNF a). Tumor necrosis factor a isa potent cytokine produced by various cell types includingmonocytes, in response to inflammation, infection, injury, andother environmental challenges. It plays a unique and pivotalrole in regulating apoptotic signaling pathways, and in the con-trol of cell proliferation and inflammation [31]. Tumor necrosisfactor a can induce cell apoptosis through the activation of acaspase cascade [32], and the downstream pathways for acti-vation of caspases, NF-kB, and other cellular responses includea variety of kinases such as p38 and JNK, and other specializedsignaling proteins [33,34]. Therefore, TNF a response triggeredby SPs and their metabolites may account, at least in part, for thecytotoxicity of monocytes. NF-kB activity, which is mediatedusing TNF a receptor associated proteins, can be blocked withIL-10 [35]. That would partially lead to the inhibition of cellviability. A previous study suggested that SPs inhibited signaltransduction in human lymphocytes ex vivo [11], and thepresent results further demonstrated that the common SPmetabolites can also inhibit signal pathways in human mono-cytes and may induce immune dysfunctions.
Results from the present study showed that the metabolismproducts of SPs may be more immunotoxic than the parentcompounds. In particular, the aldehyde derivative induced moreintensive apoptosis and greatly upregulated the secretion of IL-12P70, while the acid derivative caused the strongest inhibitionof cell viability and intensive cell apoptosis, and the highestsecretion of TNF a. As discussed previously, despite the differ-ent cyclopropane acid moieties in different SPs, all SPs having
2510 Environ. Toxicol. Chem. 29, 2010 Y. Zhang et al.
the alcohol moiety are metabolized in a similar manner toproduce the common metabolites of PBCOH, PBCHO, andPBCOOH. Therefore, for many SPs in use today, metabolismresults in intermediates with enhanced target organ toxicitysuch as immunotoxicity. Although a number of explanationsmay exist for the increased metabolite toxicity [3], the mech-anisms behind the enhanced immunotoxicity are far from clear.The action sites of SPs were thought to be related to integralproteins and phospholipids in the lipid bilayer owing to theirhigh hydrophobicity [36]. The phenoxybenzyl alcohol moietythat would further produce the common metabolites maydetermine the preferential location in the hydrophobic coreof biological membrane [37]. This suggests that the metabolitesmay be easier to move into the blood and lymph than the parentcompounds, and subsequently alter the downstream signaltransduction cascade after extracellular cytokine interactions,which ultimately induce higher immunotoxicity [11]. Anotherexplanation is that the metabolites may be the active compo-nents of the parent compounds, and the immunotoxicity inducedby parent compounds is due to their metabolites. However,much remains to be understood in relation to the molecularmechanisms of the increased toxicity.
In conclusion, the present study showed that in an in vitromodel, the common metabolites of SPs possessed increasedimmunotoxicity as compared to the parent compounds. Stron-ger cytotoxic effects by the common metabolites were found inthe monocytes, followed by increased disruptions of cytokinesecretion. A remarkable finding of the present study is, there-fore, the importance of considering the common metabolites inachieving more comprehensive health risk assessment of thissignificant class of man-made compounds.
Acknowledgement—The authors thank Pingping Shen (Nanjing University,Jiangsu, China) and Xujun He (Key Laboratory of Gastroenterology ofZhejiang Province, Zhejiang, China). The present study was supported by theNational Natural Science Foundations of China (20877071, 20837002) andthe National Basic Research Program of China (2009CB421603).
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