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Toxicology xxx (2012) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Changes in male hormone profile after occupational organophosphate exposure.

A longitudinal study
C. Aguilar-Garduño a,b , M. Lacasaña b,c,∗ , J. Blanco-Muñoz d , M. Rodríguez-Barranco c , A.F. Hernández e ,
S. Bassol f , B. González-Alzaga c , M.E. Cebrian g
a
Centre of Public Health Research (CSISP), Valencia, Spain
b
CIBER of Epidemiology and Public Health (CIBERESP), Spain
c
Andalusian School of Public Health, Granada, Spain
d
National Institute of Public Health, Cuernavaca, Morelos, Mexico
e
Department of Legal Medicine and Toxicology, University of Granada School of Medicine, Granada, Spain
f
School of Medicine of Torreon, Autonomous University of Coahuila, Torreon, Mexico
g
Center for Research and Advanced Studies of the National Polytechnic Institute (IPN), Mexico, DF, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: There is a growing concern about the endocrine effects of long-term, low-level exposure to organophos-
Received 30 May 2012 phate (OP) compounds. Studies on experimental animals have found that OP pesticides have an impact on
Received in revised form 29 October 2012 the endocrine system and a few clinical and epidemiological studies have also shown that OPs may affect
Accepted 1 November 2012
the male hormone profile, although results are inconsistent. We have evaluated the effect of exposure
Available online xxx
to OP pesticides, measured through urinary levels of six dialkylphosphate (DAP) metabolites, on male
hormone profile in 136 floriculture workers from the State of Mexico and Morelos during two agricul-
Keywords:
tural periods with different degree of pesticide exposure. Generalized estimated equations (GEE) models
Sex hormones
Pesticides
were developed and adjusted for several potential confounders, including PON1 enzyme activity, as a
Endocrine system biomarker of susceptibility, and serum levels of p,p -DDE, a metabolite of the pesticide DDT widely used
Occupational health in Mexico until 1999 for control of agricultural pests and malaria. Exposure of male floriculture work-
Endocrine disrupting chemicals ers to OP pesticides was associated with increased serum levels of follicle-stimulating hormone (FSH)
and prolactin and with decreased serum testosterone and inhibin B levels. Among all DAPs tested, only
DETP was inversely associated with luteinizing hormone (LH). Estradiol showed a marginally significant
positive trend with DEP and DETP derivatives. In conclusion, OP pesticides may have an impact on the
endocrine function because of their potential to modify the male hormone profile as a function of the
type of pesticide used as well as the magnitude of exposure.
© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The acute adverse effects of OP on the central nervous sys-

Organophosphate (OP) pesticides are non-persistent com-
pounds widely used for pest control at home or in agriculture
activities. Workers employed in agriculture-related industries are
at increased risk from pesticide exposure as confirmed by higher
burdens of pesticide metabolites in biological samples in compar-
ison to those found in subjects from non agricultural communities
(Bouvier et al., 2005; Bradman et al., 2007; Cocker et al., 2002).
Therefore, there is a growing concern about long-term, low-level
exposure to OP compounds.
∗ Corresponding author at: Escuela Andaluza de Salud Pública, Campus Universi-
tario de Cartuja, c/Cuesta del Observatorio 4, 18180 Granada, Spain.
Tel.: +34 958 027556.
E-mail address: marina.lacasana.easp@juntadeandalucia.es (M. Lacasaña).
tem lies in the inhibition of acetylcholinesterase (AChE) at nerve
endings, which leads to the accumulation of the neurotransmit-
ter acetylcholine and consequently over-stimulation of muscarinic
and nicotinic receptors (Bjørling-Poulsen et al., 2008; Ecobichon,
2001). However, little is known about chronic exposure to these
chemicals, which has been associated with a number of health
outcomes including neurocognitive effects (Baldi et al., 2001),
neurodegenerative diseases (Franco et al., 2010), certain cancers
(Bonner et al., 2010), adverse reproductive effects (Moreno-Banda
et al., 2009; Peiris-John and Wickremasinghe, 2008), respiratory
effects (Hernández et al., 2011) and endocrine disruption (Blanco-
Muñoz et al., 2010; Lacasaña et al., 2010; Meeker et al., 2006).
Experimental studies have shown that OP can act as endocrine
disrupting chemicals (EDC) because of their capacity to impair the
male sexual hormone profile (Kang et al., 2004; Rattner et al., 1986;
Sarkar et al., 2000). Furthermore, clinical and epidemiological stud-
ies conducted in men with environmental or occupational exposure

0300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tox.2012.11.001

Please cite this article in press as: Aguilar-Garduño, C., et al., Changes in male hormone profile after occupational organophosphate exposure. A
longitudinal study. Toxicology (2012), http://dx.doi.org/10.1016/j.tox.2012.11.001
 ARTICLE IN PRESS
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to OPs have also shown that these compounds may affect the male
hormone profile (Blanco-Muñoz et al., 2010; Guven et al., 1999;
Meeker et al., 2006; Padungtod et al., 1998; Recio et al., 2005). How-
ever, these studies show inconsistent results that can be accounted
for by distinct characteristics of exposure, biomonitoring of differ-
ent OP pesticide metabolites, different study design and statistical
analysis of results (Blanco-Muñoz et al., 2010; Kelce et al., 1995;
Toppari et al., 1996).
Individual vulnerability to OP toxicity depends on the lifestyle
of subjects, including smoking and dietary habits, and genetic sus-
ceptibility to these xenobiotics (Costa et al., 2005a; Lacasaña et al.,
2010). Genetic susceptibility to some OP pesticides is primarily
determined by polymorphisms in the paraoxonase-1 (PON1) gene,
which codifies for a key enzyme involved in the hydrolysis of toxic
primary-metabolites (oxon-metabolites) of OPs. There is a varia-
tion up to 40 times in PON1 activity in humans as a result of either
coding or regulatory region polymorphisms, such as PON1Q192R
and PON1C-108T (Costa et al., 2005a; Richter et al., 2009). Hydrol-
ysis of OPs containing a thiono sulfur group (such as parathion,
chlorpyrifos or diazinon) generates a chemical residue specific for
each OP compound (the ‘leaving group’ after hydrolysis) and six
dialkylphosphate (DAP) derivatives, which are non-specific for a
particular OP (Costa et al., 2005b). DAPs are ultimately eliminated
by the urine and then this matrix allows for monitoring previous
exposure to about 85% of all OP compounds (Aprea et al., 1996;
Cocker et al., 2002; Costa et al., 2005b).
On the other hand, organochlorine pesticides such as DDT have
been widely used in Mexico until 1999 for control of agricultural
pests and malaria (Yañez et al., 2002). Measurable levels of p,p-
dichlorodiphenyldichloroethene (p,p -DDE), the major and most
persistent DDT metabolite, has been reported to act like an EDC
because of its capability to impair the endocrine system (Boas et al.,
2006; Blanco-Muñoz et al., 2012). Recently, levels of p,p -DDE have
been detected in various biological samples (serum, maternal milk,
cord blood and adipose tissue) from both endemic and non endemic
Mexican populations (Barraza-Vázquez et al., 2008; Galván-Portillo
et al., 2002; Torres-Sánchez et al., 2007; Trejo-Acevedo et al., 2009).
However, at present OP pesticides are far more commonly used
than organochlorine compounds in the Mexican floriculture indus-
try.
The aim of this study was to evaluate the effect of OP pesticide
exposure, measured through urinary levels of six DAP metabolites,
on male hormone profile in floriculture workers from the State of
Mexico and Morelos (the major producers and exporters of flowers
in Latin America) during two agricultural periods (rainy and dry
seasons) with different degree of pesticide exposure and adjusting
for several potential confounders, including PON1 enzyme activity
and serum levels of p,p -DDE.
2. Materials and methods
2.1. Design and study population
A longitudinal study was conducted on male floriculture workers, with at least
6 months working in this industry, in the State of Mexico and Morelos (Mexico)
during two periods: July–October (rainy season) 2004 and December 2004 to May
2005 (dry season). These periods correspond to the two main agricultural periods in
which large quantities of pesticides are sprayed (rainy season) and where pesticides
are less heavily sprayed (dry season).
Workers were identified through employees’ records from 57 businesses in this
field. Workers performed different activities with different levels of exposure to
pesticides, from administrative task to mixing and application of these compounds.
All businesses used a traditional production system, including the regular use of
pesticides, except for one business where organic production methods were used
(n = 15 workers).
During the rainy season, a total of 143 eligible workers between 18 and 52
years of age, with at least 6 months working in this industry with no evidence
of chronic diseases (such as diabetes mellitus, liver illness, renal insufficiency or
cancer), known infertility or endocrine diseases were selected and invited to par-
ticipate in the study. All participants were informed of the general objectives and
Please cite this article in press as: Aguilar-Garduño, C., et al., Changes in male hormone profile after occupational organophosphate exposure. A
longitudinal study. Toxicology (2012), http://dx.doi.org/10.1016/j.tox.2012.11.001
procedures of the study and one hundred thirty six (95%) agreed to participate and
signed informed consent. These workers were later appointed for the administration
of a structured questionnaire and for collecting urine and blood samples. Eighty-four
(62%) workers provided biological samples again in the dry season.
The study was approved by the Ethics Commission of the National Institute of
Public Health of Mexico.
2.2. Administration of questionnaires and sample collection
Questionnaires were administered and biological samples collected one day
after pesticide application at the location where each participant worked. A struc-
tured questionnaire was administered to the floriculture workers included in
the study, containing items about sociodemographic characteristics (age, mar-
ital/cohabitant status, education and family income), anthropometric measures
(weight and height), alcohol and smoking habits, clinical and surgical history,
type of working activity in the floriculture industry and its duration (occupational
exposure), and exposure to specific pesticides or other chemicals at home (residen-
tial exposure). Additionally, another questionnaire was administered to workers
involved in mixing and loading of pesticides to ascertain the specific pesticides used
(commercial name) on the day before the collection of biological samples in both
agricultural periods. Pesticides more frequently used by the study population have
been reported elsewhere (López-Flores et al., 2009; Schilmann et al., 2010), and
reveals that the OP pesticides more often applied in both growing seasons were
omethoate, diazinon, methyl parathion and methamidophos.
In the days prior to questionnaire administration and biological sample col-
lection, each participant was instructed to self-collect a first voided urine sample
(before 8:00 a.m.) at home in a supplied container. Blood samples (10 ml) were taken
in fasting conditions, between 8:00 and 9:30 a.m., and deposited in vacutainer tubes
without anticoagulant, and later centrifuged to obtain serum. The aliquots of serum
and urine were stored at −20 ◦ C and −70 ◦ C, respectively, at the National Institute
of Public Health of Mexico (INSP), until they were analyzed.
The administration of questionnaires and collection of biological samples were
done by nursing personnel specially trained and standardized for this purpose who
did not know the main hypotheses of the study. The study was approved by the
Ethics Committee of the INSP.
2.3. Laboratory procedures
2.3.1. Measurement of hormones of the reproductive axis
The pituitary hormones (FSH, LH and prolactin), steroid hormones (testosterone
and estradiol) and inhibin B were quantified at the School of Medicine of Torreon,
Autonomous University of Coahuila (Mexico). LH, FSH and prolactin were measured
by enzyme-linked immunosorbent assay (ELISA) solid phase enzyme-amplified sen-
sitivity immunoassay, performed on microtiter plates (Biosource, Nos. KAQ1311,
KAQ0841 and KAQ1441, respectively). The limits of detection (LOD) for LH, FSH and
prolactin were 0.1, 0.15 and 7.6 mIU/ml, respectively, with inter-assay coefficients
of variation (CV) of 6.0, 8.9 and 7.1%. The intra-assay CV were 4.9, 4.2 and 4.6%,
respectively.
Testosterone was measured using the R&D Systems ELISA Kit (DE2300), which
has a sensitivity of 3.8 pg/ml and inter- and intra-assay CV of 9.3 and 7.8%, respec-
tively. Inhibin B was measured using a commercially, double antibody ELISA
(DSL-10-84100 Active inhibin B) with inter- and intra-assay CVs of 7.6% and 4.6%,
respectively, with a sensitivity of 10 pg/ml. Estradiol was also measured using a
commercial double antibody ELISA (Diagnostic Automation Inc., No. 2046z), with
inter- and intra-assay CV of 6.6 and 4.9%, respectively, and a sensitivity of 10 pg/ml.
All absorbances were measured spectrophotometrically using a Perkin Elmer
35 and Dynotech MR5000 spectrophotometer. The reference values for hormones
were provided by the laboratory, according to World Health Organization (WHO)
criteria (WHO, 1999).
2.3.2. Dialkylphosphates (DAPs) measurement
Six common DAP metabolites of OP pesticides were measured in urine samples:
dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophos-
phate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP) and
diethyldithiophosphate (DEDTP). Extraction of DAPs from urine was performed
according to the method reported by Ueyama et al. (2006). Compounds were mea-
sured by gas–liquid chromatography using a flame photometric detector equipped
with a filter isolating phosphorous emissions.
The limits of quantization (LOQ) and detection (LOD) were calculated according
to the US Environmental Protection Agency specifications (2000) and had the same
values of 50 and 22.5 mg/l, respectively, for each of the six DAPs metabolites. For non-
quantifiable values (trace amounts) half of the LOQ was assigned and for values that
were not detectable half of the LOD was assigned. For the statistical analysis, 25 mg/l
was used for the trace category and 11.25 mg/l for the non-detectable category.
The mean intra- and inter-assay CV for urinary OP metabolites under study were
between 10 and 15%, mean recoveries ranged from 102 to 119%.
Because DAP metabolites originate from more than one OP pesticide, their mea-
surement provide no specific information about the pesticide to which workers
were exposed to. However, DAPs were used as biomarkers of exposure because 85%
of OP pesticides used in floriculture may produce from one to three (in the same
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group of DMPs or DEPs) of the six DAP metabolites, and provide useful informa-
tion about the OPs exposure. Dimethyl (DMP, DMTP and DMDTP) and diethyl (DEP,
DETP and DEDTP) metabolite concentrations were converted to their molar con-
 
centrations (mmol/l) and summed to produce a single methyl ( DMP) or ethyl
( DEP) phosphate concentration and total DAPs ( DAP) concentration for each
sample (Lu et al., 2001). Metabolite concentrations were adjusted for creatinine
concentration to correct for variable urine dilutions.
2.3.3. Creatinine quantification
Urinary creatinine was quantified by colorimetry. In alkaline solution, creati-
nine reacted with picric acid to form a colored complex, in an amount that was
directly proportional to the creatinine concentration. A commercial creatinine kit
(Randox) was used for this purpose and samples were read on a VITALAB Eclipse
spectrophotometer.
2.3.4. Serum p,p -DDE levels
In order to assess the exposure to organochlorine compounds, serum levels of
p,p -DDE were measured by using gas chromatography-electron capture detection
following the protocol recommended by the US Environmental Protection Agency
(1980). p,p -DDE is the principal metabolite of the organochlorine pesticide DDT
(dichlorodiphenyltrichloroethane) and it is even more persistent than DDT in both
the environment and live organisms. Concentration of p,p -DDE was expressed on
wet basis (ng/ml). The detection limit was 0.0125 ng/ml.
For internal quality control, each serum sample was fortified with aldrin and the
average recovery was 98.15 ± 8.8%. For every 10 study samples, one sample of bovine
serum with known quantity of p,p -DDE was analyzed, with a 103.4% of recovery.
Additionally, one randomly selected sample was analyzed in duplicate in each batch
with a coefficient of variation of 4.37% for p,p -DDE.
Total lipids in serum were measured using a colorimetric method kit (Randox
Laboratories Ltd., Antrim, United Kingdom).
DAP metabolites, p,p -DDE, creatinine and total lipids were measured at Center
for Research and Advanced Studies of the National Polytechnic Institute (CINVES-
TAV), Mexico.
2.3.5. Paraoxonase-1 (PON1) activity
Serum PON1 activities toward paraoxon (POase activity), diazoxon (DZOase
activity) and phenylacetate (AREase activity) were measured as described in detail
elsewhere (López-Flores et al., 2009). Briefly, POase activity was measured using
1.25 ␮M paraoxon in absence or presence of 1 M NaCl (basal and salt-stimulated
POase activity) at 25 ◦ C. The liberation of p-nitrophenol was monitored spec-
trophotometrically at 412 nm. The enzyme activity was expressed as nmol of
p-nitrophenol/min/ml of serum. DZOase activity was assessed by monitoring the
hydrolysis of diazoxon (diazinon-O-analog) into 2-isopropyl-4-methyl-6-hydroxy
pyrimidine (IMHP) at 270 nm using 250 ␮M of diazoxon and at 25 ◦ C. Enzyme activity
was expressed as ␮mol of IMHP/min/ml of serum.
Arylesterase activity (AREase) was measured by using 1 ␮M phenylacetate as
the substrate at 25 ◦ C. Catalytic activity was determined by following the increase
of phenol concentration at 270 nm and expressed as the number of ␮mol of pheny-
lacetate hydrolyzed/min/ml of serum.
POase and AREase activities were measured at National Institute of Cadiology
“Ignacio Chavez”, Mexico, DF.
2.3.6. Statistical analysis
General characteristics of the study population were described with number of
individuals and proportions. The 2 test and Fisher’s test were used to determine
differences of general population characteristics among agricultural periods (rainy
and dry season).
The Kolmogorov–Smirnov test was applied to assess the normal distribution
of dependent variables. Thus, prior to modeling, dependent variables were all log-
arithmically (ln)-transformed and exposure variables were (log10 )-transformed to
improve normality or to reduce skewness.
Urinary DAPs, serum p,p -DDE, serum levels of sex hormones (testosterone,
prolactine, estradiol, FSH, LH, inhibin B) and PON1 activity against three differ-
ent substrates (AREase, POase and DZOase) were compared across two agricultural
periods (rainy vs. dry seasons) by means of Wilcoxon’s test. Geometric mean (GM)
and percentiles (25, 50, 75 and 95) are presented for characterizing data.
Creatinine-adjusted urinary DAP levels (untransformed) and male sexual hor-
mones levels (untransformed) were compared among the group of workers
employed in companies using conventional non-organic production and workers
employed in organic production using the non-parametric Mann–Whitney test.
Associations between individual DAP metabolites and hormone levels were esti-
mated by means of generalized estimating equation (GEE) models adjusted for both
inter and intra-individual variability and taking into account the seasonal variabil-
ity of pesticide application patterns. GEE models were also adjusted for potential
confounders, such as age, income level, educational level, body mass index (BMI),
tobacco and alcohol consumption, number of years spent as a floriculture worker,
occupational and pesticide exposure, residential pesticide exposure, living close to
industry, place of residence (State of México and Morelos), PON1 activity (AREase;
POase; DZOse), p,p -DDE, creatinine and total lipids, based on their biological plau-
sibility and on the current scientific knowledge.
Please cite this article in press as: Aguilar-Garduño, C., et al., Changes in male hormone profile after occupational organophosphate exposure. A
longitudinal study. Toxicology (2012), http://dx.doi.org/10.1016/j.tox.2012.11.001
3
Multivariate adjusted GEE models were further calculated and potential con-
founding variables were considered in the adjusted models, if they were related to
outcome, based on Wald tests with a p-value of <0.20, or if their inclusion changed
the parameter estimate for the association between OP metabolites and hormone
concentration by >10%. The adjusted models also included those variables which,
although did not fulfill this statistical criterion, were considered to be biologically
important, specifically age and BMI.
Two models were constructed for each sexual hormone. The first one considered
DAP levels as continuous variables (ln-transformed) and the second one categorized
DAP levels into tertiles, which allowed the calculation of p-trend values.
All the statistical analyses were conducted with STATA statistical software (ver-
sion 8.2, StataCorp LP, Texas) and SPSS statistical package (version 15.0, SPSS Inc.,
Chicago, IL). We rejected the null hypothesis when the p value was <0.05.
3. Results
3.1. Characteristics of the study population
The study population mostly came from Morelos (76% vs. 24%
of men from the State of Mexico). Of the workers, 70% applied or
mixed pesticides during the rainy season and 75% during the dry
season.
Socio-demographic characteristics of the study population in
both agricultural seasons are shown in Table 1. There were no sig-
nificant differences between the rainy and dry season as regards
age, BMI, tobacco and alcohol consumption, years as floriculture
worker, living close to industry and occupational pesticide expo-
sure. Approximately 80% of workers were between 20 and 49 years
old. More than 50% of the participants showed a BMI ≥ 25 kg/m2
and reported no to be current smokers, but 15% of them reported
alcohol consumption above 30 g/day. For the rainy and dry seasons,
82 and 88% of participants, respectively, reported a history of agri-
cultural work of more than 5 years. Likewise, 70 and 75% of workers
in the rainy and dry seasons, respectively, reported to mix or apply
pesticides.
Residential exposure to pesticides and organic solvent from
paint differed significantly across the two periods considered in
this study. Paint use during the dry season was more frequently
reported (58%), while pesticides were more frequently applied at
home during the rainy season (41%).
A total of 136 floriculture workers provided information and
biological samples during the rainy season, and 84 of them provided
information and biological samples again during the dry season.
Data from these 84 individuals who participated in the two seasons
were compared with data from the 52 individuals who withdrew
from the study during the dry season and no significant differences
were found for any of the general characteristics with the exception
of years spent as a floricultural worker. Moreover, there were no
differences in levels of biomarkers of exposure (p,p -DDE and DAPs),
biomarkers of susceptibility (PON1 activities) and biomarkers of
effect (FSH, LH, prolactin, testosterone, inhibin B) except for serum
levels of estradiol.
3.2. Metabolites of OP pesticides and p,p -DDE levels
More than 90% of the workers had at least one DAP metabolite
present in the urine during the rainy season against 69% of work-
ers during the dry season. DMP was the most frequent metabolite
(92.7%) found in the rainy season, followed by DMTP (68.4%), DEP
(79.4%), DETP (48.5%), DMDTP (40.5%), and finally DEDTP (10.3%).
During the dry season, DMP was again the most frequent metabo-
lite (27.4) found, followed by DMTP (22.7%), DETP (19.1%), DMDTP
(14.3%), DEP (13.5%) and DEDTP (7.2%).
Urinary levels of the six DAPs metabolites were significantly
higher during the rainy season (p < 0.01 in all cases). OP metabolites
with dimethyl moieties (DMP, DMTP and DMDTP) were detected at
higher concentrations than those with ethyl moieties (DEP, DETP
and DEDTP) in both agricultural periods. For the rainy season, DMP
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Table 1
General characteristics of the study population in both agricultural seasons.

Rainy season Dry season p-Value

n (%) n (%)

Total 136 (100) 84 (100)

State
Morelos 104 (76.5) 64 (76.2)
State of Mexico 32 (23.5) 20 (23.8) 0.962a

Age group (years)

<20 10 (7.4) 6 (7.1)
20–29 47 (34.6) 25 (29.8)
30–39 44 (32.4) 27 (32.1)
40–49 29 (21.3) 20 (23.8)
≥50 6 (4.4) 6 (7.1) 0.874a

BMI group
Normal weight (<25) 64 (47.1) 41 (48.8)
Overweight (25–30) 54 (39.7) 30 (35.7)
Obesity (>30) 18 (13.2) 13 (15.5) 0.804a

Tobacco smoke
Non-smoker 29 (21.3) 18 (21.4)
Past smoker 40 (29.4) 29 (34.5)
Current smoker 67 (49.3) 37 (44.0) 0.694a

Alcohol intake
Non drinker 29 (21.3) 17 (20.2)
Less than <30 g/day 87 (64.0) 54 (64.3)
More than ≥30 g/day 20 (14.7) 13 (15.5) 0.975a

Years spent as a floriculture worker

<5 years 25 (18.4) 10 (11.9)
5–10 years 47 (34.6) 26 (31.0)
−20 years 40 (29.4) 29 (34.5)
>20 years 24 (17.6) 19 (22.6) 0.447a

Occupational pesticide exposure

Non-pesticide mixer/applicator 25 (18.4) 12 (14.3)
Pesticide mixer/applicator 96 (70.6) 63 (75.0)
Organic agriculture worker 15 (11.0) 9 (10.7) 0.718a

Residential paint exposure

No 95 (69.9) 35 (41.7)
Yes 41 (30.1) 49 (58.3) <0.001a

Residential pesticide exposure

No 80 (58.8) 66 (78.6)
Yes 56 (41.2) 18 (21.4) 0.003a

Use of firewood at home

No 104 (76.5) 61 (72.6)
Yes 32 (23.5) 23 (27.4) 0.522a

Use of coal at home

No 132 (97.1) 76 (90.5)
Yes 4 (2.9) 8 (9.5) 0.062b
a
 test.
2
b
Fisher’s test.

derivative showed the highest concentration (geometric mean of
0.77 ␮mol/g creatinine). Levels of DMP, DMTP and DMDTP were
reduced in the dry season in about 90, 73 and 38%, respectively
(p < 0.001) and for DEP, DETP and DEDTP the reductions observed in
the dry season were 71, 50 and 25%, respectively (p < 0.01; Table 2).
During the rainy season (period with high exposure to pes-
ticides) the group of workers employed in companies using
conventional non-organic production had significantly higher
creatinine-adjusted urinary DMP and total DAP levels than the
group of workers employed in organic production. Geometric
means (GM) for DMP were 1.75 and 0.42 ␮mol/g creatinine, respec-
tively, and GM for DAP were 2.29 and 0.67 ␮mol/g creatinine,
respectively. No significant differences were observed in urinary
DAP levels among both groups during the dry season.
Finally, geometric mean of p,p -DDE was also lower for the dry
season, but this difference was not statistically significant (p = 0.37;
Table 2).
3.3. Serum male hormone profile
All serum sexual hormones levels showed significant differences
among both agricultural periods, with the exception of LH. Geomet-
ric means of FSH, prolactin and estradiol were higher in the rainy
season (3.5 vs. 2.8 IU/l, 6.3 vs. 4.7 ng/ml and 23.9 vs. 21.3 pg/ml,
respectively) while levels of testosterone and inhibin B were lower
in the rainy season compared to the dry season (4.5 vs. 7.6 ng/ml
and 63.0 vs. 88.2 pg/ml, respectively) (Table 2).
During the rainy season statistically significant differences
were found for serum hormone levels according to the kind of
agricultural production. As compared to workers using organic
methods, individuals employed at non-organic production com-
panies had significantly higher serum testosterone levels (GM 3.45
vs. 4.00 ng/ml) whereas their FSH (GM 4.21 vs. 3.47 UI/l), prolactin
(GM 7.02 vs. 6.25 ng/ml) and inhibin B (GM 210.34 vs. 54.29 pg/ml)
levels were significantly lower. However, during the dry season

Please cite this article in press as: Aguilar-Garduño, C., et al., Changes in male hormone profile after occupational organophosphate exposure. A
longitudinal study. Toxicology (2012), http://dx.doi.org/10.1016/j.tox.2012.11.001
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Table 2
Urinary dialkylphosphate levels, serum sexual hormone concentrations and PON1 activities of the study population in both agricultural seasons.

Rainy season Dry season p-Value*

N = 136 N = 84

GM SD P25 P50 P75 P95 GM SD P25 P50 P75 P95

Dialkylphosphates (␮mol/g creatinine)

DMP 0.77 13.33 0.34 0.69 1.76 14.68 0.08 0.54 0.03 0.05 0.16 1.48 <0.001
DMTP 0.22 24.45 0.07 0.16 0.46 3.79 0.06 0.54 0.03 0.04 0.11 0.68 <0.001
DMDTP 0.08 0.44 0.04 0.06 0.14 0.94 0.05 0.52 0.02 0.03 0.05 0.77 0.001
DEP 0.14 0.35 0.07 0.12 0.25 1.04 0.04 0.19 0.02 0.03 0.06 0.28 <0.001
DETP 0.08 0.59 0.04 0.06 0.11 0.85 0.04 0.12 0.03 0.04 0.06 0.22 <0.001

DEDTP 0.04 0.05 0.03 0.03 0.05 0.17 0.03 0.17 0.02 0.03 0.04 0.10 0.004
 DMP 1.50 28.97 0.62 1.26 3.44 21.04 0.27 0.90 0.09 0.26 0.77 2.24 <0.001

DEP 0.29 0.85 0.16 0.24 0.42 2.06 0.13 0.36 0.08 0.12 0.20 0.50 <0.001
DAP 2.00 29.05 0.84 1.62 4.23 23.88 0.48 0.96 0.19 0.47 1.08 2.50 <0.001

Organochlorines
p,p -DDE (ng/ml) 6.2 18.2 2.9 4.9 11.1 57.8 4.7 20.4 2.2 4.7 11.1 51.10 0.37

Sexual hormones
FSH (UI/l) 3.5 0.8 3.0 3.6 4.1 5.4 2.8 4.9 1.8 3.1 4.3 6.8 0.013
LH (UI/l) 5.6 2.7 4.4 5.7 7.5 10.1 6.0 2.5 5.1 5.8 6.8 8.7 0.446
Testosterone (ng/ml) 4.5 1.3 3.5 4.4 5.6 7.2 7.6 4.2 5.9 7.3 10.0 16.3 <0.001
Prolactin (ng/ml) 6.3 1.8 5.3 6.3 7.4 9.7 4.7 2.4 3.8 4.4 5.3 9.2 <0.001
Inhibin B (pg/ml) 63.0 123.9 29.0 55.4 129.5 375.7 88.2 60.8 61.0 93.7 130.7 219.0 0.024
Estradiol (pg/ml) 23.9 18.4 20.8 23.0 26.2 36.4 21.3 4.2 18.7 20.4 24.3 30.0 <0.001

PON1 activity
AREase (mmol/min/ml) 100 30 87 102 123 152 94 32 73 101 121 152 0.259
Basal POase (nmol/min/ml) 183 179 116 177 279 534 91 61 61 114 142 224 <0.001
Saline POase (nmol/min/ml) 417 367 250 473 718 1157 300 182 244 338 486 642 <0.001
DZOase (mmol/min/ml) 11.4 6.9 8.7 11.7 14.6 22.6 10.4 4.0 7.6 10.8 13.7 17.9 0.056

Geometric mean (GM); standard deviation (SD), selected percentiles 25th, 50th, 75th, and 95th.
*
Wilcoxon’s test.

serum levels of inhibin B were significantly higher in individuals
employed at non-organic production companies than in workers
employed in organic production (GM 95.66 vs. 45.03 pg/ml).
As regards the reference ranges of male hormonal profile
(Table 3), results indicated that for the rainy season 5.1, 2.9, 1.5
and 1.5% of subjects had levels of FSH, LH, inhibin B and estradiol,
respectively, above the reference ranges, while 5.9% of the work-
ers had levels of LH and testosterone below the reference values.
For the dry season 4.8, 1.2 and 2.4% of workers had FSH, LH and
testosterone values below the reference levels, respectively, while
32, 14.3 and 2.4% showed testosterone, FSH and LH levels above
their respective reference values.
3.4. Serum PON1 activity
As a key enzyme in the metabolic detoxification of some OPs,
serum PON1 activity was used as biomarker of susceptibility to
these chemicals in the studied participants. Geometric means for
serum POase activity (basal and salt-stimulated) were significantly
higher during the rainy season as compared to the dry season
(p < 0.001). Geometric mean of serum DZOase activity was also
higher during the rainy season than during the dry season (p < 0.05).
However, no difference was observed in serum AREase activity
between both periods (Table 2).
3.5. Associations between concentrations of male reproductive
hormones and DAP levels
The results of the GEE models for each of the six male hor-
mones studied (FSH, LH and prolactin, testosterone, inhibin B and
estradiol) and OP pesticide urinary metabolite levels are shown
in Table 4. The exposure was characterized as the sumof the
urinary concentrations of three dimethyl derivatives ( DMP:
DMP
 + DMTP + DMDTP), sum of three diethylphosphate derivatives
( DEP: DEP + DETP + DEDTP) and sum of six dialkylphosphate
  
derivatives ( DAP: DMP + DEP). These metabolites were also
categorized into tertiles. Adjusted GEE models were developed
independently for each DAP metabolite to evaluate the potential
association with serum male hormones (Fig. 1).
 GEE models showed a significant positive association of urinary
DAP with serum levels of FSH and prolactin and a negative asso-
ciation with testosterone and inhibin B (Table 4). With respect to
LH, no significant association was found, except for a reduction with
urinary
 DETP levels (Table 4 and Fig. 1).
DMP levels categorized into tertiles were positively
 and
monotonically associated with FSH (p-trend <0.002). DEP lev-
els (tertiles) also showed a significant association with FSH, but in
this case no dose–response relationship was observed (p for trend
p < 0.05). DAP levels were positively associated with FSH, but this
association was not monotonic (p-trend <0.06) (Table 4). 
 Serum prolactin levels were positively associated with DMP,
DEP, and DAP levels, showing a strong dose–response
association (p-trend <0.002). There were a significant negative
association
 between testosterone
 levels and urinary
 concentrations
of DMP, DEP and DAP. However, only DEP showed a clear
dose–response association (p-trend <0.003) (Table 4).
 Inhibin B demonstrated a monotonic negative association with
DMP levels categorized into tertiles (p-trend <0.07). However,
DEP showed this negative association when they were analyzed
as continuous variable (p < 0.05; Table 4).
GEE adjusted models for each one of the six individual DAP
derivatives are shown in Fig. 1. Serum FSH levels showed a positive
association with all DAP metabolites, although only a significant
association was found for methyl derivatives. LH levels showed
only a significant reduction with urine DETP levels. Serum prolactin
showed a positive significant association with all DAP derivatives
except DMDTP and DEDTP metabolites. All these associations were
stronger for diethylphosphate metabolites. Testosterone levels
were also inversely associated with all DAP derivatives, with DMP,
DMDTP, DEP and DETP showing statistically significant reductions

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Table 3
Sexual hormone levels and serum reference levels in the study population in both agricultural season.

n GM Min Max Reference value Below reference value (%) Above reference value (%)

Rainy season
FSH (UI/l) 136 3.5 2.1 7.5 1–5 0 5.1
LH (UI/l) 136 5.6 1.8 25.5 3–12 5.9 1.5
Testosterone (ng/ml) 136 4.5 2.0 7.5 3–9 5.9 0
Prolactin (ng/ml) 136 6.3 3.8 15.8 3–19 0 0
Inhibin B (pg/ml) 136 63.0 6.6 667.9 10–531 2.2 2.9
Estradiol (pg/ml) 136 23.9 2.0 225.1 <60 0 1.5

Dry season
FSH (UI/l) 84 2.8 0.4 44.0 1–5 4.8 14.3
LH (UI/l) 84 6.0 4.0 24.6 3–12 0 2.4
Testosterone (ng/ml) 84 7.6 1.2 27.0 3–9 1.2 32.1
Prolactin (ng/ml) 84 4.7 1.3 17.0 3–19 2.4 0
Inhibin B (pg/ml) 84 88.2 12.0 345.0 10–531 0 0
Estradiol (pg/ml) 84 21.3 15.8 31.9 <60 0 0

Geometric mean (GM).

Below reference value: percentage of individuals with hormone levels below reference value.
Above reference value: percentage of individuals with hormone levels above reference values.

Table 4
GEE models analyzing the association between urine dialkylphosphate levels and serum concentration of sexual hormones.

FSH (UI/l)a LH (UI/l)b Testosterone (ng/ml)c

ˇ p-Value CI 95% ˇ p-Value CI 95% ˇ p-Value CI 95%


ln( DMP) 0.152 0.001 (0.064; 0.240) −0.002 0.954 (−0.069; 0.065) −0.108 0.012 (−0.193; −0.023)
 1er tertil (<0.39) Ref. – Ref. – Ref. –
DMP (␮mol/g
2do tertil (0.39–1.33) 0.173 0.015 (0.033; 0.312) 0.004 0.942 (−0.101; 0.109) −0.078 0.258 (−0.214; 0.057)
creatinine)
3er tertil (>1.33) 0.233 0.002 (0.087; 0.380) 0.036 0.529 (−0.076; 0.148) −0.115 0.103 (−0.253; 0.023)
p-Trend 0.002 0.539 0.107

ln( DEP) 0.077 0.331 (−0.078; 0.233) −0.078 0.180 (−0.193; 0.036) −0.245 0.001 (−0.389; −0.102)
 1er tertil (<0.13) Ref. – Ref. – Ref. –
DEPs (␮mol/g
2do tertil (0.13–0.26) 0.258 <0.001 (0.119; 0.397) 0.055 0.302 (−0.050; 0.160) −0.161 0.016 (−0.292; −0.030)
creatinine)
3er tertil (>0.26) 0.142 0.060 (−0.006; 0.289) 0.026 0.653 (−0.086; 0.138) −0.206 0.003 (−0.342; −0.069)
p-Trend 0.043 0.599 0.003

ln( DAP) 0.154 0.004 (0.050; 0.259) −0.014 0.730 (−0.093; 0.065) −0.150 0.003 (−0.250; −0.050)
 1er tertil (<0.59) Ref. – Ref. – Ref. –
DAP (␮mol/g
2do tertil (0.59–1.80) 0.150 0.039 (0.008; 0.292) 0.028 0.598 (−0.077; 0.133) −0.056 0.412 (−0.189; 0.077)
creatinine)
3er tertil (>1.80) 0.142 0.060 (−0.006; 0.291) −0.022 0.702 (−0.134; 0.090) −0.126 0.082 (−0.268; 0.016)
p-Trend 0.057 0.716 0.080

Prolactin (ng/ml)d Inhibin B (pg/ml)e Estradiol (pg/ml)f

ˇ p-Value CI 95% ˇ p-Value CI 95% ˇ p-Value CI 95%


ln( DMP) 0.115 <0.001 (0.053; 0.178) −0.137 0.113 (−0.307; 0.033) 0.035 0.263 (−0.026; 0.095)
 1er tertil (<0.39) Ref. – Ref. – Ref. –
DMP (␮mol/g
2do tertil (0.39–1.33) 0.059 0.248 (−0.041; 0.159) −0.220 0.111 (−0.489; 0.050) 0.025 0.615 (−0.071; 0.121)
creatinine)
3er tertil (>1.33) 0.166 0.002 (0.063; 0.269) −0.258 0.070 (−0.537; 0.021) 0.004 0.941 (−0.097; 0.104)
p-Trend 0.002 0.068 0.933

ln( DEP) 0.222 <0.001 (0.116; 0.329) −0.297 0.045 (−0.588; −0.007) 0.066 0.212 (−0.038; 0.171)
 1er tertil (<0.13) Ref. – Ref. – Ref. –
DEP (␮mol/g
2do tertil (0.13–0.26) 0.162 0.001 (0.067; 0.258) −0.011 0.937 (−0.281; 0.259) 0.106 0.030 (0.010; 0.201)
creatinine)
3er tertil (>0.26) 0.256 <0.001 (0.155; 0.356) −0.098 0.496 (−0.382; 0.185) 0.063 0.222 (−0.038; 0.164)
p-Trend <0.001 0.509 0.197

ln( DAP) 0.147 <0.001 (0.074; 0.220) −0.163 0.112 (−0.364; 0.038) 0.041 0.261 (−0.03; 0.112)
 1er tertil (<0.59) Ref. – Ref. – Ref. –
DAP (␮mol/g
2do tertil (0.59–1.80) 0.131 0.010 (0.031; 0.230) −0.089 0.527 (−0.364; 0.187) 0.063 0.196 (−0.033; 0.16)
creatinine)
3er tertil (>1.80) 0.217 <0.001 (0.114; 0.320) −0.150 0.301 (−0.434; 0.134) 0.042 0.410 (−0.058; 0.143)
p-Trend <0.001 0.302 0.391

GEE models based on 215 observations: 134 belonging to the rainy season and 81 to the dry season. Dependent variables were ln-transformed.
a
Adjusted for state of residence, age, BMI, years spent as a floriculture worker, working as pesticide mixer/applicator, paraoxonase, diazoxonase and arylesterase activities,
and serum p,p -DDE levels.
b
Adjusted for state of residence, age, BMI, years spent as a floriculture worker, tobacco consumption, working as pesticide mixer/applicator, paraoxonase, diazoxonase
and arylesterase activities, and serum p,p -DDE levels.
c
Adjusted for state of residence, age, BMI, years spent as a floriculture worker, working as pesticide mixer/applicator, residential paint exposure, residential use of
pesticides, paraoxonase, diazoxonase and arylesterase activities, and serum p,p -DDE levels.
d
Adjusted for state of residence, age, BMI, years spent as a floriculture worker, working as pesticide mixer/applicator, residential use of pesticides paraoxonase, use of
firewood and/or coal at home,diazoxonase and arylesterase activities, and serum p,p -DDE levels.
e
Adjusted for state of residence, age, BMI, alcohol intake, years spent as a floriculture worker, working as pesticide mixer/applicator, residential use of pesticides,
paraoxonase, diazoxonase and arylesterase activities, and serum p,p -DDE levels.
f
Adjusted for state of residence, age, BMI„ years spent as a floriculture worker, working as pesticide mixer/applicator, paraoxonase, diazoxonase and arylesterase activities,
and serum p,p -DDE levels.

Please cite this article in press as: Aguilar-Garduño, C., et al., Changes in male hormone profile after occupational organophosphate exposure. A
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0,7 0,7

0,5 0,5

0,3 0,3
and CI 95%

and CI 95%
0,1 0,1

-0,1 -0,1

-0,3 -0,3

-0,5 -0,5

-0,7 -0,7
∑DMP DMP DMTP DMDTP ∑DEP DEP DETP DEDTP ∑DAP ∑DMP DMP DMTP DMDTP ∑DEP DEP DETP DEDTP ∑DAP

C (TESTOSTERONE) D (PROLACTIN)
0,7 0,7

0,5 0,5

0,3 0,3
and CI 95%

and CI 95%

0,1 0,1

-0,1 -0,1

-0,3 -0,3

-0,5 -0,5

-0,7 -0,7
∑DMP DMP DMTP DMDTP ∑DEP DEP DETP DEDTP ∑DAP ∑DMP DMP DMTP DMDTP ∑DEP DEP DETP DEDTP ∑DAP

E (INHIBIN B) F (ESTRADIOL)
0,7 0,7

0,5 0,5

0,3 0,3
and CI 95%
and CI 95%

0,1 0,1

-0,1 -0,1

-0,3 -0,3

-0,5 -0,5

-0,7 -0,7
∑DMP DMP DMTP DMDTP ∑DEP DEP DETP DEDTP ∑DAP ∑DMP DMP DMTP DMDTP ∑DEP DEP DETP DEDTP ∑DAP

Fig. 1. GEE models analyzing the association between the six urine dialkylphosphates and serum levels of FSH (A), LH (B), testosterone (C), prolactin (D), inhibin (E) and
estradiol (F). All GEE models were adjusted for state of residence, age, BMI, years spent as a floriculture worker working as pesticide mixer/applicator, POase, AREase and
DZOase activities, and serum p,p -DDE levels. GEE models for figures B–E were also adjusted for the following variables: (B) tobacco consumption; (C) residential paint and
pesticides exposure; (D) residential pesticides exposure and use of firewood and/or coal; (E) alcohol intake and residential pesticides exposure. The dependent variables
(serum hormone levels) were logarithmically (ln)-transformed and exposure variables (urinary DAP levels) were (log10 )-transformed.

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8 C. Aguilar-Garduño et al. / Toxicology xxx (2012) xxx–xxx
(Fig. 1). Inhibin B levels were also inversely associated with the six
individual DAP metabolites, although significant reductions were
found only for DMP and DEP. Serum estradiol showed a positive,
but not significant association with all DAPs metabolites (Fig. 1).
4. Discussion
The present study shows that exposure of male floricultural
workers to OP pesticides, as measured by urinary concentrations
of DAPs, is associated with increased serum levels of FSH and pro-
lactin and with decreased serum testosterone and inhibin B. Among
all DAPs tested, only DETP was inversely associated with LH. Estra-
diol showed a marginally significant positive trend with DEP and
DETP derivatives. To our knowledge this is the first study evalu-
ating the effect of OP pesticide urinary metabolite levels on male
hormonal profile adjusting for serum PON1 activity and p,p -DDE
concentration. Only Padungtod et al. (1998) assessed the effect of
PON1 polymorphisms but did not adjust for PON1 activity.
Exposure
 to OP  compounds, as measured by urinary concentra-
tion of DMP, DEP and DAP (either as continuous variables
or categorized into tertiles) was associated with dose-dependent
increases in prolactin and FSH, although the latter showed
 a mono-
tonic association only with DMP. DEP and DMP were
associated with decreased testosterone levels, although a signifi-
cant dose-dependent association was observed only with DMP.
Inhibin
 B showed a clearer reduction in workers with higher levels
of DMP.  data reported by Meeker et al. (2006).
In the rainy season, geometric
 mean
 levels of DMP were four
times higher than those of DEP. DMP metabolites significantly
decreased in the dry season as compared to the rainy season (ten
times lower) but were still nearly two-fold greater than DEP
metabolites. This is consistent with the patterns of pesticide use
reported by flower companies of Morelos and the state of Mexico,
which used greater amounts of OP compounds that are metabo-
lized to methyl radicals derivatives (such as omethoate, methyl
parathion and methamidophos) and lower amounts of those that
are metabolized to ethyl radical derivatives, such as diazinon and
terbufos (López-Flores et al., 2009). These results are also consis-
tent with data reported by US studies (Barr et al., 2004; Bradman
et al., 2007; Koch et al., 2002) where the urinary ratio of DMPs to
DEPs was 3/2 (Bradman et al., 2007).
Total urinary DAP levels found in this study were four times
lower than those reported for a farming population of the state
of Durango (1079.4 ppb vs. 4406.5 ppb, respectively, in the period
of high pesticide exposure for the growing season of 1997–1998;
Recio et al., 2005). OP pesticides most commonly used were very
similar in both studies (Schilmann et al., 2010), although the
metabolite profile more often detected, and their concentration,
were very different. Moreover total DAP levels observed in this
study were about 50 times higher than those reported for a Mexico-
American population in the U.S., according to data from NHANES
during the period 1999–2004 (Barr et al., 2011). Urinary levels
of DMP and DEP were higher than other methyl and ethyl phos-
phates, respectively, and DMDTP and DEDTP were less frequently
detected. This metabolite pattern has been observed in other stud-
ies (Kavvalakis and Tsatsakis, 2012).
The reduction in testosterone levels after OP pesticides exposure
was consistent with experimental studies in rats (Joshi and Sharma,
2012), “in vitro” cell cultures (Usmani et al., 2003) and with other
human epidemiologic studies (Meeker et al., 2006; Straube et al.,
1999). However, a cross-sectional study carried out by our group in
floriculture workers of the state of Morelos found a marginally sig-
nificant positive association between total urinary DAPs and serum
testosterone concentration (Blanco-Muñoz et al., 2010). This dis-
crepancy could be due to a different epidemiological design rather
Please cite this article in press as: Aguilar-Garduño, C., et al., Changes in male hormone profile after occupational organophosphate exposure. A
longitudinal study. Toxicology (2012), http://dx.doi.org/10.1016/j.tox.2012.11.001
than to differences in the type of pesticides used. While Blanco-
Muñoz et al. performed a cross-sectional study, this is a longitudinal
study with data adjusted for PON1 enzyme activities. The analysis
of longitudinal data by using GEE models and stratifying by the two
geographical areas studied (Morelos and Mexico State) showed a
negative significant association between urinary DAP levels and
serum testosterone concentration in the two groups of workers
(data not shown). This observation is consistent with other human
studies that also found a negative association between OP pesticide
urinary metabolite levels and testosterone (Recio et al., 2005).
The change in serum testosterone may be due to more than
one mechanism, and the effects of OP pesticides can be altered in
highly estrogenic environments. Sarkar et al. (2000) showed that
the simultaneous administration of OP and estradiol to rats resulted
in an attenuated increase in testosterone levels with respect to
when rats were dosed only with OP (sublethal doses of quinalphos).
Testosterone levels were even higher in rats dosed only with estra-
diol. In turn, prolactin levels increased as also occurred in this study.
Changes in serum levels of testosterone and prolactin showed
similar trends after OP pesticide exposure, although in an opposite
fashion (Fig. 1). This suggests that the initial reduction in serum
testosterone stimulates prolactin secretion at the hypothalamus
or the pituitary that in turn increases the number of LH recep-
tors in Leydig cells, thus promoting higher testosterone production
(Guven et al., 1999; Sarkar et al., 2000; Vázquez, 1984) and pre-
venting a further drop in its levels. The change pattern observed in
the present study for testosterone and prolactin is consistent with
Inhibition of AChE by OPs can increase ACh levels in the pituitary
and hypothalamus. As a result of AChE inhibition, OP may sig-
nificantly increase ACh, GABA, epinephrine, norepinephrine (NE),
dopamine (DA) and 5-hydroxytriptamine (5-HT) concentrations
(Glisson et al., 1974; Gupta et al., 1984), ultimately affecting secre-
tion of pituitary hormones (Krsmanovic et al., 1998). 5-HT and
NE can directly stimulate the release of prolactin by the pitu-
itary (Clemens et al., 1978; Pilotte and Porter, 1981). Prolactin is
a pituitary hormone that can be modulated by cholinergic ago-
nists (Findling and Tyrrel, 1991; Smallridge et al., 1991). Also,
brain cholinergic activity increases prolactin secretion (Findling
and Tyrrel, 1991).
The OP pesticide chlorpyrifos may induce gene expression
and biosynthesis of gonadotropin-releasing hormones (GnRH) “in
vitro” (Gore, 2002) and can inhibit adrenal steroidogenesis as also
do other OPs (Civen et al., 1977; Walsh et al., 2000). Inhibition of
AChE in the hypothalamus may also alter the rate of GnRH secre-
tion (Krsmanovic et al., 1998) and thus modify pituitary secretion
of LH and FSH (Fig. 2). However, variation in GnRH secretion reg-
ulates the cyclic release of FSH by the anterior pituitary to a lesser
extent than LH. The role of GnRH in the feedback of FSH secretion
has been also less clearly defined than for LH secretion (Nussey and
Whitehead, 2001; Turek, 2004).
Exposure to OP pesticides, as measured by total DAP categorized
into tertiles, was associated with reduced serum levels of inhibin B.
However, Meeker et al. (2006) found no association. In a previous
study of our group (Blanco-Muñoz et al., 2010), urinary DAP levels
were associated with a significant reduction of inhibin B, very likely
as a result of the involvement of inhibin B in the negative feedback
of FSH. In the present study, OP pesticide urinary metabolite levels
were associated with increased FSH levels, which in turn may lead
to a reduction in inhibin B (Fig. 2).
Because testosterone stimulates the production of inhibin B,
decreased testosterone levels results in a reduction of inhibin B con-
centration, which further induces FSH and LH secretion (Damstra
et al., 2002) (Fig. 2). Although this sequence of events was only
observed for FSH, the lack of the expected increase in LH could
be due to its lower half life, which is five times lesser than that of
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OP pescides
Hypothalamus
GnRH
Hypophysis
Prolacn
FSH LH
Tess
Inhibin B Sertoli cells Leydig cells
Testosterone
Spermatogenesis
Inhibion
Smulaon
Fig. 2. Mechanism of action of the hypothalamus–pituitary axis and its regulatory
feed-back loop. The normal stimulation of the hypothalamic–pituitary–testicular
axis is highlighted with green-solid lines and the inhibitory feed-back with red-
dotted lines. Hypothalamic gonadotropin-releasing hormones (GnRH) stimulate
secretion of two gonadotropins, luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) from cells in the anterior pituitary. In the testes, LH binds to
receptors on Leydig cells, stimulating testosterone synthesis and FSH promotes
spermatogenesis. Testosterone exerts a negative feedback at the hypothalamus and
pituitary, thereby suppressing LH and FSH secretion. Inhibin B inhibits the secre-
tion of the gonadotropins both directly at the pituitary and centrally, at the level of
the hypothalamus, and thus reduces serum testosterone levels. High testosterone
levels exert a suppression in inhibin B secretion. OP pesticides inhibit acetyl-
cholinesterase in hypothalamus and changes the secretion of GnRH, resulting in
altered gonadotropins secretion that indirectly affects Sertoli and Leydig cells func-
tion and spermatogenesis. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)
FSH (Vázquez, 1984), and also because LH secretion is delayed after
acute stimulation by GnRH (Damstra et al., 2002).
The positive association between OP pesticide urinary metabo-
lite levels and FSH is consistent with experimental studies
performed in rats (Sarkar et al., 2000) and with an epidemiological
study carried out in Chinese factory workers involved in the pro-
duction of methamidophos and ethyl parathion (Padungtod et al.,
1998). However, contradictory results have also been reported in
other human studies where a negative association between OP pes-
ticide exposure and serum FSH was reported (Guven et al., 1999;
Recio et al., 2005). These contradictory findings can be accounted
for by the exposure to different OP pesticides and doses in the study
of Recio et al. (2005). In turn, Guven et al. (1999) studied a small
group of men and women acutely poisoned by OP pesticides, but
not with long-term exposure.
Guven et al. (1999) found no association between OP pesticide
exposure and serum levels of LH, which is partially consistent with
the results of this study except for DETP (Fig. 1). DETP levels showed
a negative association with LH, and this finding has been previously
reported by Meeker et al. (2006) in humans exposed to chlorpyri-
fos. This compound is an OP pesticide that can be dearylated to
Please cite this article in press as: Aguilar-Garduño, C., et al., Changes in male hormone profile after occupational organophosphate exposure. A
longitudinal study. Toxicology (2012), http://dx.doi.org/10.1016/j.tox.2012.11.001
9
DETP and trichloropyridinol, or activated to chlorpyrifos-oxon and
then detoxified to DEP and trichloropyridinol (Fenske et al., 2005;
Foxenberg et al., 2011). Contradictory studies have reported a pos-
itive association between exposure to OP pesticides and serum LH
levels (Rattner et al., 1986; Rawlings et al., 1998; Recio et al., 2005).
The inconsistencies observed among these studies and our
results could be attributed to differences in usage patterns of
OP pesticides and in the magnitude of exposure. Thus, workers
involved in the manufacture of parathion (Padungtod et al., 1998)
are expected to have a higher degree of exposure than agriculture
workers. In the farmworker population of Durango (Recio et al.,
2005), the pattern of pesticide exposure also differed from our
study population as regards to urinary DAP levels and DAPs pro-
file. On the other hand, differences in the use of personal protective
equipment may also play a role. As has been shown in experimen-
tal studies, changes in the hormonal profile may depend on both
the dose and the particular OP pesticide used (Sarkar et al., 2000;
Usmani et al., 2003). Nevertheless, the combined exposure to mul-
tiple pesticides may also contribute to variation in hormone levels
(Blanco-Muñoz et al., 2010; Fernández et al., 2004; Ibarluzea et al.,
2004; Kortenkamp, 2006; Sarkar et al., 2000). Inconsistencies can
also be attributed to different statistical analysis. In our study, lon-
gitudinal data were analyzed by using GEE models adjusted for
individual variability at two different time-points as well as for
other confounders. That is, levels of DAPs and sex hormones were
adjusted for seasonality by considering data from the two time
periods studied.
As regards to enzyme activities, exposure to OP pesticides was
significantly associated with increased POase activity but not with
AREase activity. Increased POase activity was found in the rainy
season as compared to the dry season, which may be related
to the different degree of exposure between both periods. The
higher substrate concentration found during the period of greater
exposure may have led to increased POase activity, indicating an
adaptive metabolic detoxification mechanism (Browne et al., 2006;
Hernández et al., 2004). Thus, enhanced POase activity may result
in higher urinary levels of OP metabolites.
No significant differences in AREase activity were found
between the rainy and dry periods. Because PON1 activity toward
phenylacetate is not affected by the PON1192 polymorphism,
AREase has been considered to indirectly represent serum levels
of PON1 protein (Furlong et al., 2006). Therefore, the POase/AREase
ratio, which represents the variation in enzyme activity between
the two periods studied adjusted for the serum level of protein
in the study population, was also significantly higher in the rainy
season than in the dry season.
Given that p,p -DDE has been shown to have estrogenic and
antiandrogenic effects and that several studies have reported an
effect of this metabolite on testosterone levels, p,p -DDE concen-
tration was considered as a confounding factor. Moreover, this
organochlorine compound has been associated to increased total
lipid content and certain lipids, such as cholesterol, are used for
the biosynthesis of steroid hormones (Aguilar-Garduño, 2010).
One of the strengths of this study is the utilization of GEE
models for the statistical analysis. They take into consideration
the two study periods assayed and thus provide more robust-
ness and greater validity and biologic plausibility as compared to
other regression models currently used in cross-sectional stud-
ies, which only address variability between individuals but not
intra-individual variability. In turn, GEE models adjust for inter-and
intra-individual variation.
This study also has some limitations. Urine DAP measurements
do not provide specific information regarding to the particular
pesticides to which workers were exposed to. Nevertheless, we
decided to use them as biomarkers for exposure since more than
85% of the OP pesticides used are metabolized up to three out of
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10 C. Aguilar-Garduño et al. / Toxicology xxx (2012) xxx–xxx
the six DAPs measured (Fenske et al., 2005) and recent studies have
reported promisingly the use of these biomarkers in other biolog-
ical matrices such as hair or meconium (Maravgakis et al., 2012;
Tsatsakis et al., 2009, 2010). Thus, DAPs provide useful information
about OP pesticide exposure. Another limitation is that only one
urine sample was collected and analyzed from each individual and
each study period. OPs are non-persistent pesticides, for which a
single measurement represents exposure occurring 24–48 h prior
to sample collection and does not reflect exposure beyond that time
window. However, some authors have suggested that a single urine
sample could correctly classify subjects according to their exposure
level during the three months prior to sample collection (Meeker
et al., 2005).
Regarding uncertainties associated with endocrine outcomes,
LH and testosterone secretion follow diurnal and pulsating patterns
with fluctuations during the day, making it difficult to adequately
evaluate their alterations through a single determination. Although
this approach has been reliably used in population studies (Moses,
1993; Schrader et al., 1993), the effect of diurnal fluctuation was
minimized in our study by collecting all blood samples between
8:00 and 9:30 a.m. On the other hand, the floriculturists’ participa-
tion would have been lesser if several blood samples would have
been requested.
In conclusion, the results of this study suggest that OP pesti-
cides may have an impact on the endocrine function because of
their potential to modify the male hormone profile as a function
of the type of pesticide used as well as the magnitude of exposure.
Different DAPs can be generated based on the usage patterns of
OPs. The hormonal profile can be modulated by interaction with
different pesticides that act at distinct levels of the endocrine sys-
tem, such as OPs and organochlorines. There is a need for further
research to confirm and extent these findings and to elucidate their
biological and clinical relevance.
Conflict of interest
Authors declare that they do not have conflict of interest.
Acknowledgments
The authors thank the participants of this study without whom
the study would have been impossible. This study was supported
by the Consejo Nacional de Ciencia y Tecnología of Mexico, CONA-
CYT (National Council of Science and Technology), Project Number:
SALUD-2002-C01-7574 and by the Fondo Sectorial de Investigación
para la Educación SEP-CONACYT, Project Number: 49793.
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