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*yz

yMOE 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）

Abstract—Risk assessment of man-made chemicals such as pesticides are mainly focused on parent compounds, and relatively little is

known about their metabolites, especially with regard to target organ damages such as immunotoxicity. In the present study, the

immunotoxicity 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. The

aldehyde and acid derivatives were more effective than the other compounds at cytotoxicity, with inhibition of cell viability by 56.8 and

50.6% at 105

mol L1

, and induction of 8.52 and 8.81% cell apoptosis, respectively. Exposure to SPs and their metabolites also led to

changes in the secretion levels of tumor necrosis factor a （TNF a） and interleukins （ILs）, and again the metabolites showed stronger

effects than the parent compounds. The aldehyde derivative upregulated IL-12p70 level by 1.87-fold, and the alcohol and acid derivative

increased 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 SPs

clearly exerted greater immunotoxic effects tomonocytes than the intact parent compounds. Results fromthe present study suggested the

need for considering metabolites in achieving more comprehensive health risk assessment of man-made chemicals, including target

organ 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 compounds

such as pesticides are subject to chemical or biochemical

transformations, leading to the formation of metabolites. For

pesticides, both parent compounds and their metabolites may

exert toxic effects on humans and other mammals. In some

instances, the transformation products of pesticides are more

prevalent in the environment or have higher toxicities than

the parent compound [1,2]. However, in general, most risk

assessment practices focus only on parent compounds, and

relatively few cases consider pesticide metabolites [3]. For

example, at present, essentially no knowledge is available on

the immunotoxicity of pesticide metabolites.

The immune system is in a complex balance interacting with

other systems and plays a critical role in maintaining the health

of humans and animals. It consists of a complicated network of

cells and mediators, such as cytokines, to act on innate and

inducible immune functions in a highly regulated manner. Both

suppression and enhancement of immune functions by certain

chemicals is thought to exhibit potential immunotoxicity of the

chemicals. The immune system appears to be a sensitive and

complex target for pesticides [4]. In view of their widespread

use and distribution, exposure to pesticides may represent an

important cause for immune system disruptions and may result

in induced immunomodulations that endanger humans and

animals [5].

Synthetic pyrethroids （SPs）, recognized as two different

types by the absence （type I） or presence （type II） of an a-

cyano group, are among the most commonly used insecticides

[6]. The popularity of SPs is attributed to their high efficacy to

insects, low environmental mobility, and relatively low mam-

malian and avian toxicity [7]. However, an increasing number

of studies show that SPs are capable of disrupting hormonal

activities [8], causing oxidative stress [9], inducing immune

suppression [10], and inhibiting signal transduction [11]. Pyr-

ethroids are metabolized oxidatively and hydrolytically to form

a number of primary and secondary metabolites [12]. Three

intermediates, i.e., 3-phenoxybenzoic alcohol （PBCOH）, 3-

phenoxybenzaldehyde （PBCHO）, and 3-phenoxybenzoic acid

（PBCOOH）, are common to many SPs. These metabolites have

been 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 that

PBCOOH was the most frequently detected metabolite in

82%of the urine samples collected from children [16]. Previous

studies showed that some SPs displayed immunotoxicological

effects [17] and endocrine disrupting activities [8], which, if

coupled with the recent finding that metabolites of SPs possess

endocrine disrupting activities [18,19], points to a likelihood for

SP metabolites to induce immunotoxicity like SPs. In addition,

because SP metabolites are much more polar than the parent

compounds, they may be more easily absorbed and therefore

contribute to increased immunotoxicity to animals and humans.

The primary objective of the present study was to evaluate

the immunotoxicity of SPs and their common metabolites. A

well-known human monocytic cell line U937 [20] was used

as the in vitro model for the assays. The monocytes play a

significant role in the innate immune system, which secretes

cytokines such as tumor necrosis factor m （TNF a） and inter-

leukins （ILs） to take part in immune reaction. It is expected

that both the results and approaches developed in the

present study may be useful for better understanding the

Environmental Toxicology and Chemistry, Vol. 29, No. 11, pp. 2505–2510, 2010

# 2010 SETAC

Printed in the USA

DOI: 10.1002/etc.298

* To whom correspondence may be addressed

（wliu@zjut.edu.cn）.

Published online 9 July 2010 in Wiley Online Library

（wileyonlinelibrary.com）.

2505immunotoxicity of SPs and their metabolites, and other toxic

effects 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 obtained

from Xinhuo Technical Institute. Lambda-cyhalothrin （LCT,

>98%） was purchased from Danyang Agrochemicals. Struc-

tures of all these compounds are given in Figure 1. All tested

compounds were dissolved in HPLC grade ethanol （Tedia） and

kept at 4 8C in the dark as stock solutions. Other chemicals or

solvents used in the present study were of cell culture, HPLC, or

analytical grade.

Cell culture and treatments

The U937 cells, obtained from the State Key Laboratory of

Pharmaceutical Biotechnology, Nanjing University, were cul-

tured in RPMI-1640 medium （HyClone） supplemented with

10% of fetal bovine serum （FBS, HyClone） at 37 8Cina

humidified CO2 incubator （Thermo Electron） of 5% CO2 and

95% air. The culture medium was refreshed every 3 d, and

replaced with the experimental medium （RPMI-1640 contain-

ing 2% FBS） for 1 d to reduce the effect of serum before

treatment. The cells were then treated with the dosing medium

（the experimental medium along with test compound at con-

centrations of 109

–105

mol L1

） for 3 d （for cell viability

assay） or 2 d （apoptosis and cytokine analysis）. A series of test

solutions were prepared in ethanol, with the final solvent

concentrations 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 upon

the reduction of thiazolyl blue （MTT, 3-[4,5-dimethylthiazol-2-

yl]-2,5-diphenyltetrazolium bromide; Amresco）. Cells in an

exponential growth status were seeded in 96-well plates at a

density of 5104

cells mL1

for pretreatment of 24 h, and then

the medium was changed to the dosing medium containing test

solutions at a range of concentrations. After 3-d exposure,MTT

solution （5mgmL1

phosphate-buffered saline [PBS]） was

added into wells, followed by incubation at 37 8C for 4 h.

The medium was then removed from the wells, and 150ml

DMSO was added per well. After mixing on a micro-mixer for

10min, the absorbance was measured at a wavelength of

570 nm with background subtraction at 650 nm using a Bio-

Rad Model 680 microplate reader （Bio-Rad Laboratories）. The

treatments were all repeated at least three times. The results

were expressed in the relative viability, which was the ratio of

each exposure group over the vehicle control.

Analysis of cell apoptosis

In the early stage of apoptosis, changes occur at the cell

surface such as translocation of phosphatidylserine [21], which

can be analyzed by using Annexin-V-Fluorescein and Proidium

Iodide （PI）. The SPs and their metabolite-induced cell apoptosis

were 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 an

initial concentration of 5104

per well were incubated with

the vehicle control or test solutions at the concentration

of 106

mol L1

in 6-well plates for 48 h. After harvest and

washing twice with cold PBS, cells were resuspended in 100ml

Annexin-V-FLUOS labeling solution （containing 2ml Annexin-

V-FLUOS labeling reagent and 2ml PI） and incubated for 15min

at room temperature in the dark. The final samples were

analyzed 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 evaluating

chemical exposure effects on the immune system [22]. To

further investigate the molecular mechanisms of toxicity, the

effects of SPs and their metabolites on cytokine secretion

were measured. Cells were cultured in 24-well plates with

the vehicle control or 106

mol L1

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 regulator

IL-12p70 were measured by commercial enzyme-linked immu-

nosorbent assay （ELISA） kits （Cusabio） according to the man-

ufacturer’s instructions. Each measurement was repeated at

least four times.

Statistical analysis

The results were presented as meanSD and tested for

statistical significance by analysis of variance （ANOVA） using

SPSS 16.0. Differences were considered statistically significant

when 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 to

investigate the responses of U937 cell line to SPs and their

metabolites. From the dose–response relationships, the test

compounds displayed an inhibitory effect on U937 viability

in a concentration-dependent manner （Fig. 2）. The viabilities of

O

OH

O

O

O

O

HO H H

3-phenoxybenzoic alcohol

PBCHO PBCOOH

O

O

Cypermethrin, CP

Cl

Cl

OCN

O

O

Cl

OCN

Lambda-cyhalothrin, LCT

F3C

O

O

OCN

d-cyphenothrin, d-CPN

H3C

H3C

O

O

O

H3C

H3C

O

O Cl

Cl

O

Permethrin, PM

d-phenothrin, d-PN

Type I SPs Type II SPs

The Metabolites

PBCOH

3-phenoxybenzaldehyde 3-phenoxybenzoic acid

H

Fig. 1. Chemical structures of synthetic pyrethroids （SPs） and their

metabolites.

2506 Environ. Toxicol. Chem. 29, 2010 Y. Zhang et al.U937 were significantly inhibited at 106

or 105

mol L1

for

all the compounds. Among the five different SPs, CP （type II）

was more toxic than the other SPs at 106

mol L1

（p<0.05）.

At 105

mol L1

, PM, d-PN, CP, LCT, and d-CPN decreased

cell viability to 90.0, 73.2, 67.0, 76.6, and 65.3%, and the

difference between PM and the other four SPs was statistically

significant （p<0.05）. The three metabolites also caused inhib-

ition of cell growth within the range of 108

–105

mol L1

.

Moreover, PBCHO and PBCOOH were much more toxic than

the parent compounds and PBCOH （p<0.001 for PBCHO and

p<0.05 for PBCOOH） at 105

mol L1

, with cell growth

inhibited by 56.8 and 50.6%, respectively. The results showed

that the metabolites induced suppression of cell viability stron-

ger than the parent compounds.

Induction of U937 cells apoptosis

As the inhibitory responses in cell viabilitymay be attributed

to arrest of cell cycles and induction of apoptosis, the Annexin-

V- FLUOS staining kit was used to determine the effects of SPs

and their metabolites at 106

mol L1

on U937 cell apoptosis.

The results showed that the numbers of early apoptotic cells

in the bottom right quadrant increased after exposure to SPs,

suggesting that SPs were able to induce visible early apoptosis

of U937 cells （Fig. 3）. No significant difference existed between

the test groups and the negative control in the percentage of

necrotic cells. The percentages of cells stained as Annexin-

V/PI （living cells）, Annexin-Vþ/PI （early apoptotic

cells）, and Annexin-Vþ/PIþ （necrotic cells） are presented

in Table 1. On average, PM, d-PN, CP, LCT, and d-CPN

treatments resulted in 4.88, 7.49, 9.50, 8.51, and 7.53% apop-

0.3

0.6

0.9

1.2

** **

C

**

0.3

0.6

0.9

1.2

** *

A

D

**

**

B

*

**

0.3

0.6

0.9

1.2 E

*

**

F

** ** ** ** **

0 1E-91E-81E-71E-61E-5

0.3

0.6

0.9

1.2 G

Concentration of SPs and their metabolites （mol L

-1

）

Fold （cell proliferation relative to vehicle control）

**

**

**

**

**

0 1E-9 1E-8 1E-7 1E-6 1E-5

**

H

** **

**

**

Fig. 2. The effects of synthetic pyrethroids （SPs） and theirmetabolites on the viability ofU937 cell lines.TheU937 cellswere exposed to a series of concentrations

of Permethrin （PM） （A）, d-phenothrin （d-PN） （B）, Cypermethrin （CP） （C）, Lambda-cyhalothrin （LCT） （D）, d-cyphenothrin （d-CPN） （E）, 3-phenoxybenzoic

alcohol （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 meanSD of at least three independent assays （

indicates p<0.05, and

indicates

p<0.01, compared to negative control by analysis of variance [ANOVA]）.

Fig. 3. Evaluation of apoptotic cells by the Annexin-V staining assay. The

U937 cells incubatedwith vehicle control （A）or106

mol L1

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 flow

cytometer. 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–IV

represent 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 2507totic cells, respectively, while the negative control group had

only 3.75% apoptotic cells. Type II SPs appeared to have

induced more early apoptosis than type I SPs. The metabolites

displayed similar effects of inducing cell apoptosis, and

PBCOH, 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 of

causing the same or more intensive apoptosis than their parent

compounds in U937 cells.

Alteration of cytokine secretion

Synthetic pyrethroids and their metabolites may not only

induce cytotoxicity, but also alter immune functions through the

alteration of cytokines such as TNF a and ILs that participate in

complex interactions with cell viability in immune cells. The

levels of IL-6, IL-10, IL-12p70, and TNF a in U937 monocytes

were determined using ELISA kits.With respect to ILs, only the

IL-12p70 level was altered by SPs and their metabolites. As

shown in Figure 4A, the secretion of IL-10 decreased slightly in

the test groups of CP, d-CPN, PBCOH, and PBCOOH, but the

differences were not significant. Moreover, no significant dif-

ferences were observed in the concentrations of IL-6 among the

different treatments （data not shown）. In addition, the levels of

IL-12p70 were not significantly affected after PM, CP, and LCT

treatments, 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 negative

control, which was significantly higher （p<0.05） than the

parent compounds. PBCOOH also increased the level of IL-

12p70 （ p<0.01）, although the increase was not statistically

significant 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 for

d-CPN （p<0.05） （Fig. 4C）. Permethrin, LCT, and PBCOH

induced similar levels of TNF a production, ranging from 4.95-

to 5.88-fold increases relative to the negative control. These

increases were also higher than PBCHO （p<0.05） （1.98-fold

increase 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 increase

induced by PBCOOH was significantly higher than all the

parent compounds （ p<0.05）. Overall, the results fromcytokine

analysis showed that the common metabolites were capable of

inducing similar or more intensive effects on cytokine secretion

than the parent compounds. These results also implied that the

Table 1. Percentage of apoptotic cells induced by synthetic pyrethroids

and their metabolites using Annexin-V staining assaya

Compound Quadrant I Quadrant II

b

Quadrant III

c

Quadrant IVd

Control 0.09 1.14 95 3.75

PM 0.03 1.2 93.9 4.88

d-PN 0.15 1.6 90.8 7.49

CP 0.01 1.51 89 9.5

LCT 0 1.97 89.5 8.51

d-CPN 0.04 1.33 91.1 7.53

PBCOH 0.04 1.02 91 7.98

PBCHO 0.03 1.47 90 8.52

PBCOOH 0 0.94 90.3 8.81

a

See Figure 2 for acronym key; PI¼proidium 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.

0.0

0.4

0.8

1.2

1.6

2.0 B

B

A

A b b

bc

b

bc

*

**

**

*

PBCOOH PBCHO PBCOH d-CPN d-PN LCT CP PM control

Fold （IL-12p70 secretion relative to solvent control）

 Control     Parent Compounds     Metabolites

0.0

0.2

0.4

0.6

0.8

1.0

1.2

A

A

A

A

c

*

*

*

Control    Parent Compounds    Metabolites

Fold （IL-10 secretion relative to solvent control）

control PM CP LCT d-PN d-CPN PBCOH PBCHO PBCOOH

*

0

2

4

6

8

10

Control    Parent Compounds    Metabolites

C

* B

A

A

bc

bc

ac

ac

ac

**

*

**

*

Fold （TNF-α secretion relative to solvent control）

control PM CP LCT d-PN d-CPN PBCOH PBCHO PBCOOH

**

**

Fig. 4. Assessment of cytokine secretions of cell culture supernatants. The

U937 cells were cultured with vehicle control or 106

mol L1

test solutions

for 48 h, and secretions of interleukin-6 （IL-6）, interleukin-10 （IL-10） （A）,

interleukin-12p70 （IL-12p70） （B）, and tumor necrosis factora（TNFa）（C）in

culture supernatantsweremeasured by enzyme-linked immunosorbent assay

（ELISA）.

indicates p<0.05, and

indicates p<0.01, relative to each

solvent control. Different lowercase letters indicate a significant difference

（p<0.05） between the parent compounds and their metabolites （a for

PBCOH; b for PBCHO; c for PBCOOH）.Different capital letters above error

bars indicate a significant difference （p <.05） between three metabolites,

while the same letter indicates no significant difference. See Figure 2 for

acronym key.

2508 Environ. Toxicol. Chem. 29, 2010 Y. Zhang et al.levels of IL-12p70 and TNF a in monocytes may be sensitive

endpoints for the evaluation of immunotoxicity of SPs and their

metabolites.

DISCUSSION

With the widespread use of pesticides, more comprehensive

risk assessment by considering their environmental metabolites

is imperative. Although limited studies previously showed

immunotoxicity of SPs using in vivo and in vitro models, the

present study demonstrated for the first time, to our knowledge,

that the common metabolites of SPs were capable of inducing

similar or more intensive immunotoxic effects than the parent

compounds.

In mammals, SPs are rapidly metabolized to less lipophilic

and more readily excreted metabolites [19]. For instance, the

elimination was nearly complete within 5 d of exposure formost

SPs following inhalation exposure, while the majority of the

dose was eliminated in the first 1 to 2 d following oral exposure

of 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 hours

after exposure depending on chemical structures. Studies

show that SPs can be metabolized in liver microsomes, hepatic

cytosol, serum, and small intestinal microsomes [24,25]. The

most important metabolism of most SPs occurring in liver

microsomes is cleavage of the central ester linkage, which

produces a cyclopropane acid and an alcohol moiety （Fig. 5）.

The alcohol moiety is then hydroxylated to produce PBCOH

that is further oxidized to PBCOOH using PBCHO [26]. Sub-

sequently, these metabolites undergo conjugation processes to

produce glucoronides of the carboxylic acid or sulfates of the

phenols, which are excreted in the urine. In the natural environ-

ment and higher plants, SPs are also metabolized or degraded to

form these common metabolites （[27]; http://ace.ace.orst.edu/

info/extoxnet/pips/pyrethri.htm）.

Evaluation of cell growth and apoptosis on the target cells

after exposure suggested that most of the parent compounds

inhibited cell viability and induced monocyte apoptosis, imply-

ing that SPs possessed cytotoxicity to the monocytic cells. This

finding was in agreement with some previous in vitro and in

vivo studies on SPs [28,29], as well as studies showing that

permethrin and deltamethrin increased apoptotic or necrotic

cell death in thymocytes [29,30]. Among the three metabolites,

PBCHO and PBCOOH significantly inhibited the U937 cell

growth within the concentrations of 108

to 105

mol L1

,

showing that the metabolites possessed much higher toxicity

than the parent compounds. The metabolites further displayed

similar or more intensive apoptosis than the parent SPs. Both

observations clearly suggested that the SP metabolites were

capable of causing higher cytotoxicity than the parent SPs in

monocytes.

Assay of cytotoxicity alone may not be adequate to show

pesticide-induced immunotoxicity, because cytokines also play

a paramount role in mediating cell–cell communication of

inflammatory and immune responses. Measurement of immune

responses of cytokine secretions is, therefore, an important

aspect in defining pesticide immunotoxicity. Analysis of the

effects of SPs and their metabolites on cytokine stimulation

showed that exposure to PBCOH and PBCOOH resulted in

greater disruption of cytokines of monocytes than the other

compounds. Although no obvious effect was noted on the

secretion of IL-10 and IL-6, specific metabolites induced more

intensive effects on the secretion of TNF a and IL-12p70 than

the parent compounds. These results suggested that the common

SP metabolites were capable of altering immune functions in

addition to inducing cytotoxicity in human monocytes. The

current understanding of interactions between cytokines is still

not clear, and therefore more research is needed to further

investigate the underlying mechanisms for these effects.

Monocytes are known to protect the body from a series of

pathogens and xenobiotics by releasing cytotoxic and proin-

fiammatory substances （e.g., TNF a）. Tumor necrosis factor a is

a potent cytokine produced by various cell types including

monocytes, in response to inflammation, infection, injury, and

other environmental challenges. It plays a unique and pivotal

role in regulating apoptotic signaling pathways, and in the con-

trol of cell proliferation and inflammation [31]. Tumor necrosis

factor a can induce cell apoptosis through the activation of a

caspase cascade [32], and the downstream pathways for acti-

vation of caspases, NF-kB, and other cellular responses include

a variety of kinases such as p38 and JNK, and other specialized

signaling proteins [33,34]. Therefore, TNF a response triggered

by SPs and theirmetabolitesmay account, at least in part, for the

cytotoxicity of monocytes. NF-kB activity, which is mediated

using TNF a receptor associated proteins, can be blocked with

IL-10 [35]. That would partially lead to the inhibition of cell

viability. A previous study suggested that SPs inhibited signal

transduction in human lymphocytes ex vivo [11], and the

present results further demonstrated that the common SP

metabolites can also inhibit signal pathways in human mono-

cytes and may induce immune dysfunctions.

Results from the present study showed that the metabolism

products of SPs may be more immunotoxic than the parent

compounds. In particular, the aldehyde derivative induced more

intensive apoptosis and greatly upregulated the secretion of IL-

12P70, while the acid derivative caused the strongest inhibition

of cell viability and intensive cell apoptosis, and the highest

secretion of TNF a. As discussed previously, despite the differ-

ent cyclopropane acid moieties in different SPs, all SPs having

O

OH

O

O

O

O

HO

H

H

O

O X

Y

OCN

O

O X

Y

O

Type I SPs Type II SPs

H

OH

X

Y

O

O

X

Y

O

OH-

Z

Ester cleavage

Hydroxylation and conjugation

PBCOH

PBCHO

PBCOOH

 Conjugation

Oxidization

Oxidization

Fig. 5. Metabolism of synthetic pyrethroids （SPs） in mammals. PBCOH¼

3-phenoxybenzoic alcohol; PBCHO¼3-phenoxybenzaldehyde; PBCOOH¼

3-phenoxybenzoic acid.

Immunotoxicity of pyrethroid metabolites Environ. Toxicol. Chem. 29, 2010 2509the alcohol moiety are metabolized in a similar manner to

produce the common metabolites of PBCOH, PBCHO, and

PBCOOH. Therefore, for many SPs in use today, metabolism

results in intermediates with enhanced target organ toxicity

such as immunotoxicity. Although a number of explanations

may 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 integral

proteins and phospholipids in the lipid bilayer owing to their

high hydrophobicity [36]. The phenoxybenzyl alcohol moiety

that would further produce the common metabolites may

determine the preferential location in the hydrophobic core

of biological membrane [37]. This suggests that the metabolites

may be easier to move into the blood and lymph than the parent

compounds, and subsequently alter the downstream signal

transduction cascade after extracellular cytokine interactions,

which ultimay induce higher immunotoxicity [11]. Another

explanation is that the metabolites may be the active compo-

nents of the parent compounds, and the immunotoxicity induced

by parent compounds is due to their metabolites. However,

much remains to be understood in relation to the molecular

mechanisms of the increased toxicity.

In conclusion, the present study showed that in an in vitro

model, the common metabolites of SPs possessed increased

immunotoxicity as compared to the parent compounds. Stron-

ger cytotoxic effects by the common metabolites were found in

the monocytes, followed by increased disruptions of cytokine

secretion. A remarkable finding of the present study is, there-

fore, the importance of considering the common metabolites in

achieving more comprehensive health risk assessment of this

significant class of man-made compounds.

Acknowledgement—The authors thank Pingping Shen （Nanjing University,

Jiangsu, China） and Xujun He （Key Laboratory of Gastroenterology of

Zhejiang Province,Zhejiang,China）.The present studywas supported by the

National Natural Science Foundations of China （20877071, 20837002） and

the National Basic Research Program of China （2009CB421603）.

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