Levels of Dichlorodiphenyltrichloroethane (DDT) and Hexachlorocyclohexane (HCH) pesticide residues in Sera of Breast Cancer Patients and Controls: A Case-Control Study in Ile-Ife, Southwestern Nigeria

Levels of Dichlorodiphenyltrichloroethane (DDT) and Hexachlorocyclohexane (HCH) pesticide residues in Sera of Breast Cancer Patients and Controls:
A Case-Control Study in Ile-Ife, Southwestern Nigeria
IJTEH
Levels of Dichlorodiphenyltrichloroethane (DDT) and
Hexachlorocyclohexane (HCH) pesticide residues in
Sera of Breast Cancer Patients and Controls: A Case-
Control Study in Ile-Ife, Southwestern Nigeria
*Yemisi Tosin Awe1, Adedeji Ayodeji Onayade2, Mosudi Babatunde Sosan3, John Adekunle
Oyekunle4, Olusegun Isaac Alatise5, Olalekan Olasehinde6, Adewale Adisa7
1
Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife, Nigeria
2
Institute of Public Health, Obafemi Awolowo University, Ile-Ife, Nigeria
3
Department of Crop Production and Protection, Obafemi Awolowo University, Ile-Ife, Nigeria
4
Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
5,6,7
Department of Surgery, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria
Serum levels of Dichlorodiphenyltrichloroethane (DDT) and Hexachlorocyclohexane (HCH) of
breast cancer patients and controls were compared with a view to determining association
between exposure of organochlorine pesticides (OCPs) and breast cancer. Fifty breast cancer
patients and fifty age-matched control women were recruited from the Obafemi Awolowo
University Teaching Hospitals Complex, Ile-Ife, Nigeria. Questionnaires were administered to
collect information on demography and essential breast cancer risk factors. Five millilitres of
blood was collected from each participant and the serum was analysed for DDT and HCH using
Gas Chromatography coupled with Electron Capture Detector. The results showed that almost all
case women had no identifiable risk factors for breast cancer. The median DDT levels among case
and control women were 11.87 ppb and 6.395 ppb, respectively. The levels of δ-HCH among case
and control women were 5.82 ppb and 0.00 ppb while that of γ-HCH were 10.84 ppb and 0.00 ppb,
respectively. This study confirmed exposure to OCPs among the studied population and revealed
significantly higher levels (p≤0.05) in case women than controls, thereby, suggesting that
exposure to OCPs may be a significant risk factor for breast cancer in Nigeria.
Key words: Pesticides; Organochlorine; Dichlorodiphenyltrichloroethane (DDT); Hexachlorocyclohexane (HCH); Breast
Cancer
INTRODUCTION
Ever since Rachel Carson raised public awareness about
the effects of DDT use on our health and environments in
her book, Silent Spring in 1962 (Carson, 2002), DDT and
other organochlorine pesticides have been regarded as
synthetic chemicals of environmental health concern.
Organochlorine pesticides (OCPs) are chlorinated
hydrocarbons that are used extensively in agriculture and
for vector control (UNEP, 2003) and have been linked to a
wide range of human health hazards ranging from acute
health impacts, such as headaches, dizziness and nausea
to chronic impacts such as cancer, reproductive harm and
endocrine disruption (Guo et al., 2017; Giulivo et al., 2016;
Toft, 2014). Many of these effects are due to the ability of
OCPs to alter the levels of certain hormones, enzymes,
growth factors and neurotransmitters (Kleanthi et al.,
*Corresponding author: Yemisi Tosin Awe, Institute of
Ecology and Environmental Studies, Obafemi Awolowo
University, Ile-Ife, Nigeria. Tel: +2347068419799; Email:
aweyemi4u@yahoo.com; Co-Authors Email:
2
aonayade@yahoo.com; Tel: +2348037184015;
3msosan@oauife.edu.ng Tel: +2348034041583;
4oyekunle@oauife.edu.ng; Tel: +2348035673017;
5segunalatishe@gmail.com; Tel: +2348033859387;
6lekanolasehinde@yahoo.com; Tel: +2348060716670,
7wadisc@yahoo.com; Tel: +2348033889425;
International Journal of Toxicology and Environmental Health
Vol. 3(1), pp. 027-035, July, 2018. © www.premierpublishers.org. ISSN: 1822-424X
Research Article

Awe et al. 028
2008). For example, they are considered to be endocrine
disrupters because they are weakly oestrogenic or anti-
oestrogenic in experimental assay (Mostafalou and
Abdollahi, 2017). Dichlorodiphenyltrichloroethane (DDT)
and Hexachlorocyclohexanes (HCHs) are examples of
OCPs. International Agency for Research on Cancer
(IARC) has classified DDT as Group 2A carcinogens
(probably carcinogenic to human), HCH except γ-HCH as
Group 2B (possibly carcinogenic to human) while γ-HCH
was classified as Group 1 (carcinogenic to humans) based
on sufficient evidences on experimental animals (IARC,
2013).
The number of new cases of breast cancer among women
represents 25% of all cancer cases and almost quadrupled
from one region to another throughout the world (IARC,
2013) and presently the most common form of cancer
among women globally (Balekouzou et al.,2016). In
Nigeria, the prevalence and burden of breast cancer is
under-reported. Adetifa and Ojikutu (2009) reported that,
between 1960 and 1980, prevalence of cervical cancer
was 19.9% while breast cancer was 11.2% but between
1981 and 1995, breast cancer has taken over the lead with
25.7% while cervical cancer followed closely with 22.7%.
This is not due to reduction in cervical cancer but an
increase in the incidence of breast cancer (Adisa, 2013).
In addition, Abdulkareem (2009) reported a geometric
increase in frequency of breast cancer in Nigeria over the
years and observed it to be the most common cancer in
both sexes.
The most frequently reported risk factors of breast cancer
are mainly related to reproductive characteristics of
women (Kleanthi and Andreas, 2009). In general,
prolonged or increased exposure to oestrogen is
associated with increased risk of breast cancer and vice
versa. Therefore, factors that increase the number of
menstrual cycles (early menarche, nulliparity, late onset of
menopause) are associated with increased risk while
factors that decrease the number of ovulations (long
lactation, high parity) can be protective (Schottenfeld and
Fraumeni, 2006). Other known risk factors are genetic
(BRCA1 and BRCA2 gene mutation), exogenous
oestrogen (oral contraceptives and hormone replacement
therapies), radiation, alcohol consumption, obesity and
higher educational and socioeconomic status (Kleanthi
and Andreas, 2009). However, all these factors are
thought to account for less than 50% of cases of breast
cancer that actually occur, and more than 50% of cases
are regarded as being unexplained (Brody and Rudel,
2003).
In order to determine the unexplained risk factors of breast
cancer, there has been continued interest in the role of
organochlorines and other environmental pollutants (Calle
et al.,2002). Moreover, the observation that the rising
incidence of breast cancer is aligned with the chronological
patterns of release of organochlorine compounds in the
environment; and attribution of a small fraction of breast
cancer to known risk factors make it reasonable to
investigate whether chemicals that persist in the
environment are potential risk factors for breast cancer
(Calle et al., 2002). Although, many studies have
investigated the relationship between organochlorine and
breast cancers, the results have remained inconclusive
(Kleanthi and Andreas, 2009; Schottemfeld and Fraumeni,
2006; Brody and Rudel, 2003; Calle et al., 2002).
In spite of the fact that organochlorine pesticides
importation, sales and use have been banned in Nigeria
since 2004 when the country ratified the Stockholm
Covention on Persistent Organic Polluatants (POPs) in
which she was a signatory in 2001, there are evidences of
continued usage and presence in the ecosystem. For
example, Adeyemi et al. (2011) evaluated concentration of
OCP residues in water samples of Lagos Lagoon and
found that the mean concentration of organochlorine
pesticides detected were higher than the European
Community allowable residual limit for individual OCPs in
drinking water in 37.3% of samples analyzed. Also, Sosan
et al. (2015) examined DDT and HCH residues in foodstuff
sold in markets in Ile-Ife, southwestern Nigeria and
concluded that organochlorine pesticides are still being
used to the extent that they were detected in cowpea
grains and dried yam chips in concentration above the
European Union- Maximum Residue Limits (EU-MRLs).
These two recent studies showed that Nigerians are still
greatly exposed to OCPs. Despite several studies on
relationship between OCPs and breast cancer in
developed countries, the continuing exposure of Nigerians
to these chemicals and the increased rate of breast cancer
incidence in Nigeria, there is scarcity of data on the
relationship between organochlorines and breast cancer
incidence in the country. Thus, this present study was
undertaken to comparatively evaluate the residue levels of
DDT and HCH in the serum samples of diagnosed breast
cancer patients and control subjects. This will provide
baseline information on the possible association between
these OCPs and breast cancer incidence in Nigeria.
MATERIALS AND METHODS
Study Design and Study Population
The study was a case-control study to assess association
between exposure to OCP and breast cancer. Fifty cases
and 50 controls were recruited from Obafemi Awolowo
University Teaching Hospitals Complex (OAUTHC).
Cases were women diagnosed with breast cancer, while
controls were patients (women) without breast cancer or
other diseases that are known to be related with OCPs,
such as nervous system damage, birth defects, infertility
or other reproductive problems and damage of the liver,
kidneys, lungs and other body organs. Written consent
was obtained from each of the participants and those who

Int. J. Toxicol. Environ. Health 029
were unwilling to give consent were excluded from the
study and the protocol was approved by Ethical and
Research Committee of OAUTHC, Ile-Ife, Nigeria with
protocol number ERC/2016/08/08.
Data were collected from cases and controls with a pre-
designed questionnaire that sought information on the
socio-demographic characteristics and risk factors for
breast cancer that were categorized into reproductive
history, lifestyle factors and family history of breast and
ovarian cancer. Information on the reproductive history
included age at menarche, age at first birth, parity,
menopause status, duration of lactation (months) and age
at menopause. Lifestyle factors included alcohol, smoking,
and use of exogenous hormone (contraceptives). The
demography information included age, marital status,
education, occupation and place of residence.
Five milliliter (5 mL) of blood was collected from each
participant and dispensed into labeled plain
decontaminated bottle after proper cleansing of the
venipuncture site with a swab soaked in methylated spirit
and transported immediately to the laboratory. In the
laboratory, the serum was separated by centrifugation and
frozen at -20oC in decontaminated labelled plastic vial until
analysis.
EXTRACTION AND ANALYSIS PROCEDURES
Extraction
A 1 mL of each serum sample was extracted by the
denaturation of the serum protein using 1%Na tungstate
and 1 N H2SO4, the precipitate was washed thrice with 0.1
N H2SO4 and the filtrate containing OCPs residues was
extracted using dichloromethane as the solvent. The
solution of the filtrate and DCM was shaken continuously
for about 10 min. The resulting extract was taken through
a clean-up regime.
Clean-up
A Parked column of about 15 cm by 2 cm was packed first
with glass wool and then with about 5 g of activated silica
gel. Also, about 2 g of anhydrous sodium sulphate was
placed at the top to absorb any water in the extract. Pre-
elution was done with about 15 mL of DCM, without
exposing the sodium sulphate layer to air, so as to prevent
the drying up of the silica adsorbent and breaking of the
packed column. The extract was allowed to run through
the capillary column with about 30 mL of the extract
solvent, dichloromethane. The eluate was collected and
evaporated to dryness under a stream of analytical grade
nitrogen (99.99%). The dried eluate was reconstituted with
1 mL n-hexane and levels of OCPs in it were profiled using
a Gas Chromatography coupled with an Electron Capture
Detector (GC-ECD).
𝐃𝐢𝐥𝐮𝐭𝐢𝐨𝐧 𝐟𝐚𝐜𝐭𝐨𝐫 =
𝐅𝐢𝐧𝐚𝐥 𝐕𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐄𝐱𝐭𝐫𝐚𝐜𝐭
𝐖𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐝𝐫𝐢𝐞𝐝 𝐜𝐥𝐞𝐚𝐧−𝐮𝐩 𝐞𝐱𝐭𝐫𝐚𝐜𝐭
……… (1)
Gas Chromatography (GC) Analysis
Analyses were performed on an Agilent GC-7890A
equipped with Electron Capture Detector (ECD).
Accurately measured 1 μg/L reconstituted eluate was
injected into the GC on a splitless basis at 250oC with the
aid of a microsyringe. This was then carried into the of DB
17 (30 m x 250 μm x 0.25 μm) column at a flow rate of 2
mL/min using Helium as carrier gas and Nitrogen as the
make-up gas. The initial temperature of the oven was
150oC which increased to 280 °C at 6 °C/min. The total run
time was 23 minutes. The residues of OCPs were
determined by comparing the peak areas of the samples
and the calibration curve of the standards. Gas
chromatographic analysis of the samples was carried out
at the Nigerian Institute of Oceanography and Marine
Research (NIOMR) Central Laboratory, Victoria Island,
Lagos, Nigeria.
Quantification of the OCPs
To get the final concentration of each analyte or total
analytes, the formular below was used
Final concentration =
GC result of analyte from chromatography x dilution factor
Volume of Sample taken for extraction
……… (2)
Quality Control Measures Adopted
Since no certified pesticide reference materials were
available during the course of this study, recovery analysis
was performed in order to evaluate the precision and
efficiency of the analytical procedures using standard
addition method. A 2 mL sample of serum was divided into
two. One part was spiked with 10 ppm standard mixture
consisting of organochlorine insecticides of interest
namely: γ-HCH and δ-HCH, and p,p'-DDT. These were
thoroughly mixed, while the other (control) part was left
unspiked. The sample were taken through the
simultaneous extraction and clean-up protocols as
enumerated in the earlier experimental steps. Also, 10 mL
of the standard 1000 mg/L mixture of the OCPs, in spectra
grade n-hexane, was put into a clean, oven-dried sample
bottle. This was dried at ambient temperature by passing
a stream of high purity nitrogen gas through it, and the
residue reconstituted using 2,2,4 – trimethylpentane. With
the aid of microsyringe, 1.0 μL of each of the spiked,
unspiked (control) and standard mixture was injected into
the GC column for GC-ECD analysis. The recoveries of
OCPs were determined by comparing the peak areas of
the OCPs after spiking with those obtained from the
evaporated standard residues. The percentage recovery
was evaluated from the relationship:
%R =
𝐴′−𝐴
𝐵
…………… (Formula 3)

Awe et al. 030
where A' is the amount of an OCP in the spiked sample, A
is the amount of OCP in the unspiked sample and B is the
amount of OCP used for spiking.
Data Analysis
All data were analysed and interpreted using the Statistical
Package for Social Sciences (SPSS Version 20). For the
questionnaires, descriptive statistics was used to
characterize the study population and to examine case-
control differences. Risk factors were compared between
cases and control using Chi-square Test for independence
categorical variables. Normality test was conducted for
residues of DDT, γ-HCH and δ-HCH using Shapiro-Wilk
test, the results revealed that the data are not normally
distributed (DDT, Z=6.888 and p-value=0.000; δ-HCH,
Z=4.616, p-value=0.000; δ-HCH, Z=4.373, p-
value=0.000). Wilcoxon (Mann-Whitney U test) and
Kruskal-Wallis H tests were conducted to examine
whether differences exist in the median values between
cases and controls, and of the parameters by the
explanatory variables including age group, place of
residence, occupation, parity and lactation period
RESULTS
Validation of Analytical Procedures Adopted
The analytical methods adopted in this study were tested
for its reliability by measuring its percentage recovery. The
percentage recovery values of 84.68% of δ-HCH to
99.21% of p, p'-DDT presented in Table 1 fell within the
70-110% as the acceptable range for recovery by the EU
Guidelines for evaluating accuracy and precision of a
method indicating that the procedures used for the
assessment of OCPs in this work were efficient and
reliable.
Table 1: Percentage Recovery for Organochlorine
Pesticides (OCPs)
Oganochlorine
Pesticides
Amount
(ppm) Used
For Spiking
Mean
Amount
(ppm) of
OCPs
Recovered
Percentage
Recovery
α-HCH 10 9.116 91.16 ± 5.81
γ-HCH 10 9.320 93.20 ± 3.63
δ-HCH 10 8.468 84.68 ± 3.76
p, p' – DDT 10 9.921 99.21 ± 3.88
Analysis of Questionnaire for Demography, some Risk
Factors associated with Breast Cancer and Past
Exposure to Pesticides
The socio-demographic characteristics (age, occupation,
place of residence, level of education, marital status) of
participants are as presented in Table 2. The cases and
controls were comparable with respect to these
characteristics.
The mean age of 46 years and 50 years for case and
control women, respectively showed no significant
difference (p ≤ 0.05). The majority of the women were
artisan/trader (61%), followed by civil servant (23%) while
the rest were either farmers or dependants. For 84% of the
women, place of residence was in the town while the
remaining 16% resided in city. Thirty six percent (36%) of
the participants had tertiary education, followed by
secondary (31%) and then primary and none education.
Result of the chi-square analyses showed no significant
differences of frequency distribution of these variables
between cases and controls (p ≤ 0.05).
All the breast cancer risk factors such as menopause
status, age at menopause, menarche, parity, family history
of breast cancer, contraceptive use, and smoking showed
no significant difference between cases and controls
except for alcohol drinking (Table 3). The control women
were more likely to have taken alcohol than the case
women. Although, not significantly different, the case
women were more likely to give birth to more children and
have higher duration of lactation.
Table 2: Social-Demographic Characteristics of Breast
Cancer Patients (Cases) and the Controls Attending
OAUTHC, Ile-Ife, 2016/2017
Characteristics Case
(n = 50)
Freq
Control
(n=50)
Freq
Total
(n = 100)
Freq
Statistica
l Indices
Age group (years)
<40
≥40
Age range (years)
Mean (SD) in years
12
38
27 – 92
50 (14)
12
38
27 – 95
46 (13)
24
76
t = 1.365
df = 48
p = 0.175
Occupation
Artisan/Trading
Farming
Civil Service
Dependents
31
2
11
6
30
5
12
3
61
7
23
9
X2
= 2.346
df = 3
p = 0.504
Place of Residence
City
Rural
5
45
8
42
13
87
X2
= 0.796
df = 1
p = 0.375
Marital Status
Married
Single/Divorced/Wid
owed
38
12
43
7
81
19
X2
= 1.624
df = 1
p = 0.20
Education
None
Primary
Secondary
Tertiary
6
10
16
18
8
9
15
18
14
19
31
36
X2
= 0.371
df = 3
p = 0.946

Table 3: Breast Cancer Risk Factors of Breast Cancer
Patients (Cases) and the Controls Attending OAUTHC, Ile-
Ife, 2016/2017
Characteristics Case
(n = 50)
Freq
Control
(n=50)
Freq
Total
(n = 100)
Freq
Statistical
Indices
Menopause Status
Pre-menopausal
Post-menopausal
23
27
29 (58)
21 (42)
52 (52)
48 (48)
X2
= 1.442
df = 1
p = 0.230
Age at Menopause
(years)
<45 years
≥45 years
(n=27)
7
20
(n=21)
3
18
(n=48)
10
38
X2
= 0.970
df = 1
p = 0.325
Mean Age (years) at
Menarche ±SD 15 ±2 16 ±2
t = -2.388
df = 48
p = 0.749
Mean Age (years) at
First Birth ±SD in 24 ±4 25 ±4
t = -0.488
df = 48
p = 0.702
Parity
Nulliparity
1-3 Children≥ 4
Children
3
15
32
1
24
25
4
39
57
X2
= 4.520
df = 2
p = 0.104
Cumulative Duration
of Lactation (Months)
0
<50
50-100
>100
4
15
20
11
1
23
19
7
5
38
39
18
X2
= 4.3
df = 3
p = 0.222
Contraceptive Use
Yes
No
11
39
13
37
24
76
X2
=0.219
df=1
p = 0.640
Alcohol
Yes
No
3
47
24
26
27
73
X2
= 22.374
df= 1
p = 0.00
Smoking
Yes
No
0
50
1
49
1
99
X2
= 1.010
df=1
P = 0.315
The median concentration of total DDT and two isomers of
Hexachlorocyclohexane (HCH) namely γ-HCH and δ-HCH
detected in the blood samples of breast cancer patients
and controls are presented in Table 4.
Dichlorodiphenyltrichloroethane (DDT) was detected in all
the samples (100% of the participants), δ-HCH in 58%
while γ-HCH in 47% of the participants. In this research,
the term DDT was used to refer to the sum of p, p'-DDT
and its metabolites (DDD and DDE), although these
metabolites were almost insignificant because they were
detected in only few of the samples; 6% for DDE and 4%
for DDD, and in very low concentrations. The median
concentrations of these three compounds were
significantly higher in cases than in the controls (Table 4).
The entire study population was analysed to determine if
variables, such as age, place of residence, occupation,
parity, lactation were associated with organochlorine
pesticide levels as presented on Table 5 using Mann-
Whitney test for two-groups variables and Kruskal-Wallis
test was used for variables with more than two groups. The
median concentrations of the three compounds were not
significantly different among groups of each variables
except for median concentration of δ-HCH between people
who live in city and town (p-value = 0.0278
Table 4: Median Concentration of DDT and HCH in Serum
Samples of 50 Case women and 50 Control women
Attending OAUTHC, Ile-Ife, 2016/2017
OCP
Pesticides
% Detection Median
Conc.
(ppb)
Mann-
Whitney
(U-Test)
P-Value
DDT
Case women
Control
women
100
100
11.87
6.40
3.957 0.0001
δ-HCH
Case women
Control
women
70
46
5.82
0.00
3.457 0.0005
γ-HCH
Case women
Control
women
60
34
10.84
0.00
2.996 0.0027
Table 5: Median Levels (ppb) of DDT and HCH among
100 case and control women, according to age, place of
residence, occupation, parity and lactation.
Variable DDT δ-HCH γ-HCH
Age
<40
≥40
Mann-Whitney U
Test
10.72
9.68
U=0.448
P-value=0.6542
3.61
3.79
U=0.625
P-value=0.5321
0.00
0.00
U=-1.094
P-value=0.2742
Place of
Residence
Semi-urban
City
Mann-Whitney U
Test
13.66
9.15
U=1.004;
P-value=0.3152
6.96
2.93
U=0.2.200
P-value=0.0278
10.94
0.00
U=0.1.328
P-value=0.1843
Occupation
Artisan/Trading
Farming
Civil service
Dependants
Kruskal-Wallis H
Test
11.0
13.66
6.81
6.97
H=2.377
P-value=0.4979
3.05
6.31
3.80
4.25
H=1.173
P-value=0.7594
6.9
10.94
0.00
0.00
H=1.130
P-value=0.7697
Parity
Nulliparity
1-3 Children
≥ 4 Children
Kruskal-Wallis H
Test
8.59
8.67
11.00
H=1.784
p-value=0.4098
2.165
0.00
3.88
H=2.446
p-value=0.2943
0.00
0.00
7.09
H=2.593
p-value=0.2735
Cumulative
Lactation
0 Months
1-50 Months
51 -100 months
>100 Months
Kruskal-Wallis H
Test
6.89
10.32
7.33
14.69
H=7.024
P-value=0.0711
4.33
3.36
2.89
7.64
H=2.574
P-value=0.4621
0.00
0.00
0.00
18.01
H=6.192
P-value=0.1026

Awe et al. 033
DISCUSSION
The mean age of breast cancer patients was 50 ± 14 years
and this implies that women in this study may likely be at
risk of breast cancer right from their fourth and fifth decade.
This finding agrees with the reports of, Adisa (2013),
Afolayan et al (2012) and Adetifa and Ojikutu (2009), that
the disease occurs at least a decade earlier among
Nigerian women with majority occuring in pre-menopausal
women with the peak age in the 4th and 5th decade. This
could be regarded as one of the challenges of breast
cancer in Nigeria.
In this study, case women and controls were essentially
comparable with respect to the well-known risk factors for
breast cancer such as age at menarche etc. Even in some
instances, the breast cancer patients presented lesser
risks. For instance, the case women had higher parity and
longer period lactation compared to the control, although
not significantly different. This is at variance with most
studies that reported nulliparity or low parity and short
period of lactation as risk factors of breast cancer
(Lankenau Medical Centre, 2011; Kleanthi and Andreas,
2009; McPherson et al., 2002). Another reproductive factor
considered is age at first birth. From literature, it has been
estimated that women who had their first child early
enough (on or before age 20) have reduced risk of breast
cancer as a result of early full differentiation of mammary
gland cells (Wei-Chu et al. 2000).
None of the participants (both cases and controls) had
family history of breast and ovarian cancer. Alcohol
consumption and smoking as risk factors of breast cancer
were poorly represented in this study among the case
women in that just a few had taken alcohol before or
smoked before, though it is not impossible that they are
secondary smokers. In fact, the number of control women
who had taken alcohol before was found significantly
higher than that of case women. In addition, less than 30%
of the case and control women had used hormone
medication for birth control (contraceptive) one or more
time before. Since just few of the case women and higher
number of the control women had used contraceptive
before, contraceptive cannot be adjudged to be a risk
factor in the population studied.
From the result obtained on the known risk factors of
breast cancer (age, reproductive history, contraceptive
use, alcohol and smoking, family history of breast cancer),
this study established that most case women had no
identifiable risk factors. It is therefore reasonable to
conclude that the known risk factors for breast cancer in
the women studied account for very few of the cases. This
agrees with the report that the known risk factors are
thought to account for less than 50% of cases of breast
cancer that actually occur (Brody and Rudel, 2003)
thereby, corroborating the suggestion made by Calle et al
(2002) that it is reasonable to investigate whether
environmental chemical that persists in the environment
are potential risk factors for breast cancer.
Median levels of the three organochlorine pesticide
compounds (DDT, γ-HCH and δ-HCH) were significantly
higher in breast cancer patients than the control. This
suggests a positive association between these
compounds and the risk of breast cancer. This positive
association between DDT and breast cancer has been
reported by many earlier epidemiological studies (Cohn et
al., 2007; Charlier et al., 2003; Hoyer et al., 2000) but
inconsistent with Gammon et al. (2002) and Romieu et al.
(2000) that suggested that DDT exposure cannot be linked
to increased breast cancer risk. However, researchers
from the California-based Public Health Institute and
Mount Sinai School of Medicine in New York based on
their findings from a five-decade study that DDT exposure
in utero was found to predict a four-fold increase in breast
cancer risk and that many American women heavily
exposed in uterus have not yet reached 50 years,
concluded that it is too soon to decide that DDT exposure
is unrelated to breast cancer (Cohn et al., 2015).
The result of significantly higher median levels of γ-HCH
and δ-HCH also revealed that the median concentrations
of γ-HCH (Lindane) and δ-HCH in cases are greatly higher
than that of controls. Aside the significant higher median
levels, residues of the two compounds were found more
frequently in the breast cancer patients than controls. This
finding agrees with the report of Mussalo-Rauhanaa et al.
(1990) who found residues of β-HCH (an isomer of γ-HCH
and δ-HCH) more frequently and higher in mean than that
of controls. However, this recent study is not consistent
with some other findings that found insignificant higher
concentration of HCHs in breast cancer patients (Li et al.,
2006; Zheng et al., 1999)
The inconsistent conclusion of several studies on the
association of OCPs and breast cancer risk in relation to
the present study can be attributed to several factors which
include difference in sample size, use of different biological
specimens (blood/adipose tissue), type of control women
(free of tumor or benign tumor) and possible individual
variation in the rate of metabolism of organochlorines.
Highest median levels of all compounds (though not
significantly different) found in farmers may be due to
occupational exposure. Lindane (γ-HCH) marketed under
the name Gamalin 20EC®, Capsitox 20EC®, Kokotine
20EC® has been for a long time popular household trade
name in most agricultural communities of Nigeria for insect
pest control in cocoa (Olufade et al., 2014). Higher levels
found among those that reside in city (significant only for
δ-HCH) may be as a result of non-occupational exposure
due to excessive use of some organochlorine pesticides to
preserve food consumed in the cities.

CONCLUSION
The evidence to date concerning the association between
organochlorine pesticides exposure and risk of breast
cancer is not entirely consistent. This study however found
that exposure to some organochlorine pesticides (DDT, γ-
HCH, δ-HCH) may increase women risk of breast cancer.
In addition, this work has shown that exposure to
environmental pollutants is a major risk factor of breast
cancer because most of the breast cancer patients present
no identifiable known factors of the disease. Notable is the
fact that, although, organochlorine pesticides have been
banned in Nigeria, there is an indication that some of these
pesticides are still in use as at the time of this study. This
was suggested because of the presence of higher
concentration and higher frequency of parent compound
(DDT) than its metabolites (DDE and DDD) in the blood
samples.
Although, this study was limited by small sample size, the
population selection methods was refined by age-
matching controls to address the sample size limitation
and increase the power of these findings. This study has
provided for the first time information on the possible
association between breast cancer and organochlorine
pesticides in Nigeria and also provided useful information
on levels of these compounds in the studied population.
ACKNOWLEDGEMENT
The moral and huge financial support of the father of the
corresponding author, Mr. Kehinde Martins Awe is greatly
acknowledged. Also, efforts of all the members of staff of
Surgical Research Office, OAUTHC, Ile-Ife, Nigeria (Drs.
O. Olajide, O. Taiwo, O. Akinwande, I. Babayomi, Mr. O.
Olalude and Mrs. O. Odebode) for the use of their
laboratory and other assistance during the study are
appreciated. Equally, efforts of all the medical doctors of
OAUTHC, Eleyele branch, Ile-Ife, Nigeria who also
facilitated blood sample collection from the study
participants are acknowledged.
FUNDING: This research received no external funding. It
is the research project for the award of M.Sc.
(Environmental Control and Management) of the
corresponding author,
CONFLICTS OF INTEREST: The authors declare no
conflict of interest
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Accepted 6 July 2018
Citation: Awe YT, Onayade AA, Sosan MB, Oyekunle JA,
Alatise OI, Olasehinde O, Adisa A (2018). Levels of
Dichlorodiphenyltrichloroethane (DDT) and
Hexachlorocyclohexane (HCH) pesticide residues in Sera
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provided the original author and source are cited.

APPENDIX
Figure 1: A sample chromatogram for one of the case samples
Figure 2: A sample chromatogram for one of the control samples

Levels of Dichlorodiphenyltrichloroethane (DDT) and Hexachlorocyclohexane (HCH) pesticide residues in Sera of Breast Cancer Patients and Controls: A Case-Control Study in Ile-Ife, Southwestern Nigeria

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