Environmental Toxicology
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 ?ve 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, cyto?ow, 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 ef?cacy 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 ?nding 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
signi?cant 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
humidi?ed 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 ?nal 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 ?nal samples were
analyzed on a ?ow cytometer （Becton Dickinson）.
Measurement of cytokine secretion
Assessment of cytokine, the molecules in response to reg-
ulating various processes including immunity, in?ammation,
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 proin?ammatory 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 signi?cance by analysis of variance （ANOVA） using
SPSS 16.0. Differences were considered statistically signi?cant
when p value was less than 0.05 or 0.01.
RESULTS
Inhibitory responses in cell viability
The MTT assay for cell vitality was ?rst 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 signi?cantly inhibited at 106
or 105
mol L1
for
all the compounds. Among the ?ve 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
signi?cant （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 signi?cant 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 ?ow
cytometer. The x axis represents increasing Annexin-V ?uorescence
（relative light unit）, and the y axis represents increasing Proidium Iodide
（PI） ?uorescence （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 signi?cant. Moreover, no signi?cant 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 signi?cantly 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 signi?cantly 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
signi?cant 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 signi?cantly 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 signi?cant 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 signi?cant difference （p <.05） between three metabolites,
while the same letter indicates no signi?cant 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 ?rst 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 ?rst 1 to 2 d following oral exposure
of humans or animals （[23]; http://www.atsdr.cdc.gov/toxpro-
?les/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
?nding 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 signi?cantly 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
in?ammatory and immune responses. Measurement of immune
responses of cytokine secretions is, therefore, an important
aspect in de?ning 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, speci?c 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-
?ammatory substances （e.g., TNF a）. Tumor necrosis factor a is
a potent cytokine produced by various cell types including
monocytes, in response to in?ammation, 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 in?ammation [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 ?nding of the present study is, there-
fore, the importance of considering the common metabolites in
achieving more comprehensive health risk assessment of this
signi?cant 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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