Exposure to Organochlorine Pesticides is Independent Risk Factor of
Hepatocellular Carcinoma: a Case-Control Study
Benhua Zhao1, Heqing Shen2, Feng Liu2, Sheng Liu1, Jianjun Niu3, Fei Guo1, Xueli
Sun4
Supplementary Information
This part include the methods for Interview, Database Setting, Serological
Biomarkers and HCC diagnosis, Serum Sample Collection and Storage, PAH-albumin
Adducts Measurement, Sample Preparation and Chemical Agents, Instrument
Condition for OCP Analysis, Calibration and Quantification, Quality Control/Quality
Assessment; the results listed in Table S1 (The correlation of the investigated OCPs in
serum in the cohort), Table S2 (Univariate logistic analysis of the HCC risk factors of
OCPs) and Table S3 (Multivariate logistic regression models for HCC risk assessment
of OCPs exposure).
Interview. Before their work, all the investigators were trained how to use the
questionnaire and to be sure they know the detail of each question written down in a
manual. During the training, mimic interviews were done and only the qualified
investigators were allowed to involve the work. Although it is hard to hide the
information from them, the study designers did not tell the investigators who is the case
and who is the control. The designers also kept the main study hypotheses in secret.
With the permission of the interviewee, a living recording was done in case of the
review for the questionnaire. After each interview, the investigators were asked to
check the uncertain answers immediately.
Database Setting. The finished questionnaires were handed in an inspector in the
same day. The inspector checked the logical problem and code of the questionnaire.
The problematic questionnaires were returned to the investigators for reviewing and the
passed ones were input the computer by the workers using version 3.1 of the EpiData
Software (EpiData Association; Odense, Denmark). The other group of workers
checked the data in the computer and did the primary analysis in case of the coding and
logical mistakes. At the end of the cohort interview, 10% case and control interviewees
from the cohort in random were re-interviewed by phone for credibility checking of the
investigation. After all of the checks passed, the database was established for the further
use.
Serological Biomarkers and HCC diagnosis. Alanine aminotransferase (ALT),
aspartate aminotransferase (AST), α-fetoprotein (AFP), hepatitis B surface antigen
(HBsAg), antibody hepatitis C virus (anti-HCV) were measured in all the blood
samples. Any individual met one of the four situations, i.e., (1) ALT > 45 IU/mL and
AST > 40 IU/mL, (2) AFP > 20 ng/mL and HBsAg or anti-HCV with positive result, (3)
first degree relatives had hepatocirrhosis and (4) family medical history of HCC, the
followed ultrasonography (USG) epigastrium test was done. The suspected HCC cases
were further diagnosed by computerized tomography (CT), digital subtraction
angiography (DSA), cytological test and biopsy. The diagnostic criteria of HCC are (1)
biopsy and at least two positive results of the three instrumental tests of CT, USG
epigastrium and DSA, or (2) serum α-fetoprotein (AFP) > 400ng/mL combing with one
positive result of the three instrumental tests.
Serum Sample Collection and Storage. With the permission, 10 mL blood
sample was taken from each patient by the nurse on an empty stomach. 5 mL blood
with sodium citrate and the other 5 mL blood free of the anticoagulant were put into the
vacuum tubes separately. The blood with anticoagulant was stored at -70oC for the
other use. The blood without anticoagulant was centrifuged at 3000 g for 8 minutes. 1.5
mL serum was taken for OCP analysis and the rest clot was stored for the other use.
They all stored -70 oC until the further use.
PAH-albumin Adducts Measurement. For detecting PAH-albumin adducts, the
albumin sepreated from 4ml plasma (Autrup et al., 1999; Sun et al., 2001) was first
extracted by using Sep-Pak C18 solid phase extraction (SPE) column, and then it was
purified with HPLC (Waters, USA) with conditions of C18 reversed-phase column
and elution program of methanol from 30% to 100%. The target compound
dihydroxyepoxy benzopyrene (BPDE) was detected by a fluorescence detector.
Sample Preparation and Chemical Agents. The serum sample preparation for
OCP analysis was modified from the US EPA method (Sundberg et al. 2006). Simply,
added urea into 0.5 mL defrosted serum and the final concentration of urea was 8 M for
denaturing the protein. Then the activated HLB SPE column (Waters, USA) was
applied for OCP extraction. After washing and elute, the extract was blowed to dryness
with mild N2 and in the final the solvent with 20ng/mL polychlorinated biphenyl (PCB)
congener PCB-82 was used to re-dissolve the extract up to 50 μL. 2μL sample was
injected into gas chromatography-mass spectrometer (GC-MS) for each analysis run.
Methanol, hexane, dichloromethane are all high performance liquid chromatography
grade purchased from TEDIA, America. Guaranteed grade urea was bought from
Sangon Biotech (Shanghai) Co., Ltd, China. Before the use, all the glass wares were
washed by water with detergent powder, then dried and dipped with 10% hydrogen
chloride solution over night. After the dryness again, heated them at 450 oC for 4.5
hours. For quantification, the mixture standard of pesticides (β-HCH, γ-HCH, p,
p’-DDE, p, p’-DDD), the recovery indicators 2, 4, 5, 6-tetrachloro-m-xylene and
PCB67, and the isotope label standard 13C-PCB82 were bought from AccuStandard.
Instrument Condition for OCP Analysis. The selected OCPs were measured by
gas chromatograph-mass spectrometer of Agilent 7890A/5975C equipped with a 30 m
× 0.25 mm i.d. × 0.25 μm film thickness DB-5MS capillary column (J&W Scientific,
USA). The used carry gas was helium with flow rate 1 mL/min. The oven temperature
program was started at 60 oC, programmed at 30 oC/min to 200 oC, 5 oC/min to 220 oC,
then 1 oC/min to 230 oC, and finally programmed at 35 oC/min to 300 oC and held 6 min.
The mass spectrometer worked at SIM mode with mass range m/z 50-500 and scan
frequency 3.3 s-1. The other parameters were: ion source temperature 230 oC, transfer
line 280 oC, quatrapole temperature 150 oC and the voltage multiplier 1700 V.
Calibration and Quantification. The working standard solutions were at
concentrations of 1, 5, 10, 20 and 50 ng/mL in hexane. The two characteristic masses
and the retention time together were used for qualification. One of the two masses
was used as calibration mass and the ratio of the peak area of the target compound
versus to the peak area of 13C-PCB82 (internal standard) was applied in quantification.
Quality Control/Quality Assessment. The target compound was locked with the
characteristic masses and the retention time. The criteria of the quantification were
peak area bigger than the three times signal/noise ratios and the two times blank
samples, where pure water (CascadaTM AN, Pall, USA) instead of serum was applied in
the sample preparation. The regressed calibration curve (peak area ratio set as Y and
standard solution concentrations as X) all reached correlation coefficient more than
0.999. The limit of detections (LOD) of the target compounds were calculated based on
the blank samples: p, p’-DDT (m/z: 235, 165) 0.008 ng/mL, p, p’-DDD (m/z: 235, 165)
0.007 ng/mL, p, p’-DDE (m/z: 318, 246) 0.006 ng/mL, o, p’-DDT (m/z: 235)
0.012 ng/mL; m/z for HCH isomers were 181 and 219 with LODs α-HCH 0.022 ng/mL,
β-HCH 0.013 ng/mL, γ-HCH 0.04 ng/mL and δ-HCH 0.083 ng/mL. The SPE spike
recoveries in water ranged from 71.6% to 101.1% and in serum ranged from 69.3% to
105.4%. The serum samples from the case subjects and the matched control ones were
assayed in the same batch, and the SPE recovery difference between them was not
found. The final quantification of the measurement was based on isotope dilution
method. Solvent blank, water blank and standard samples were run during every 10
samples. The reported values were the blank values subtracted data.
References:
Sun C.-A., Wang L.-Y., Chen C.-J., Lu S.-N., You S.-L., Wang L.-W., et al. Genetic polymorphisms of
glutathione
S-transferases
M1
and T1
associated with susceptibility to
aflatoxin-related
hepatocarcinogenesis among chronic hepatitis B carriers: a nested case–control study in Taiwan.
Carcinogenesis 2001: 22: 1289-94.
Autrup H., Daneshvar B., Dragsted L.O., Gamborg M., Hansen M., Loft S., et al. Biomarkers for
exposure to ambient air pollution-comparison of carcinogen-DNA adduct levels with other exposure
markers and markers for oxidative stress. Environ Health Perspect 1999: 107: 233-8.
Sundberg S.E., Ellington J.J., and Evans J.J. A simple and fast extraction method for organochlorine
pesticides and polychlorinated biphenyls in small volumes of avian serum. J Chromatogr B 2006: 831:
99-104.
Table S1 The correlation of the investigated OCPs in serum in the cohort, where
p
< 0.05 and p < 0.01:
-HCH
-HCH
-HCH
-HCH
p, p’-DDT
p, p’-DDE
o, p’-DDT
p, p’-DDD
-HCH
-HCH
-HCH
-HCH
p, p’-DDT
p, p’-DDE
o, p’-DDT
p, p’-DDD
1
0.222
0.245
0.354
0.378
0.156
0.491
0.187
1
0.612
0.273
0.424
0.333
0.231
0.432
1
0.523
0.416
0.198
0.326
0.467
1
0.421
0.471
0.271
0.168
1
0.333
0.111
0.367
1
0.300
0.222
1
0.281
1
Table S2 Univariate logistic analysis of the HCC risk factors of OCPs, where
OCPs’ concentrations lower than the 4th quartile in the control group were set as
the low exposure group and the concentrations bigger than the 4th quartile were
set as the high exposure group:
Compound Exposure Case (%)
-HCH
-HCH
-HCH
-HCH
p, p’-DDT
p, p’-DDE
o, p’-DDT
p, p’-DDD
*
<2.02
≥2.02
<13.43
≥13.43
<1.06
≥1.06
<1.04
≥1.04
<43.09
≥43.09
<10.56
≥10.56
<2.11
≥2.11
<0.91
≥0.91
236 (68.4)
109 (31.6)
229 (66.4)
116 (43.6)
243 (70.4)
102 (29.6)
249 (72.2)
96 (27.8)
179 (51.9)
166 (48.1)
197 (57.1)
148 (42.9)
256 (74.2)
89 (25.8)
247 (71.6)
98 (28.4)
Control (%) 2
721 (75)
240 (25)
721 (75)
240 (25)
721 (75)
240 (25)
721 (75)
240 (25)
721 (75)
240 (25)
721 (75)
240 (25)
721 (75)
240 (25)
721 (75)
240 (25)
p-Value AOR (95%CI)*
5.35
0.021
1.27 (1.05-1.53)
9.15
0.002
1.35 (1.12-1.63)
2.54
0.111
1.18 (0.97-1.44)
1.08
0.298
1.11 (0.92-1.36)
62.39 0.000
2.06 (1.72-2.45)
38.2
0.000
1.78 (1.49-2.12)
0.05
0.818
1.03 (0.84-1.27)
1.38
0.239
1.14 (0.93-1.39)
AOR was the adjusted odd ratio for age, BMI, occupation, education and marital status
Table S3 Multivariate logistic regression models for HCC risk assessment of
OCPs exposure, where age, sex, education and diabetes were set as the covariates:
Risk Factor
-HCH
-HCH
-HCH
-HCH
p, p’-DDT
p, p’-DDE
o, p’-DDT
p, p’-DDD
i
0.22
0.90
0.08
0.10
0.74
0.86
0.21
0.12
SE(i)
0.43
0.24
0.31
0.81
0.09
0.14
0.11
0.44
Wald 2
0.25
14.57
0.07
0.02
7.90
36.34
3.72
0.07
p-Value
0.66
0.00
0.80
0.90
0.01
0.00
0.05
0.79
AOR
1.24
2.45
1.08
1.11
2.08
2.35
1.24
1.12
95%CI
0.53~2.89
1.54~3.89
0.59~1.97
0.23~5.43
1.74~2.50
1.78~3.11
0.99~1.54
0.47~2.68