Journal of Exposure Science and Environmental Epidemiology (2010) 20, 310–319

r 2010 Nature Publishing Group All rights reserved 1559-0631/10

www.nature.com/jes

Biomarkers of exposure to combustion by-products in a human population

in Shanxi, China

ZIAD NAUFALa, LI ZHIWENb, LI ZHUb, GUO-DONG ZHOUa, THOMAS McDONALDa, LING YU HEa,
LAURA MITCHELLc, AIGUO RENb, HUIPING ZHUc, RICHARD FINNELLc AND KIRBY C. DONNELLY a

a
Department of Environmental and Occupational Health, Texas A&M Health Science Center, School of Rural Public Health, College Station, Texas, USA
b
Institute of Reproductive and Child Health, Peking University Health Science Center, Beijing, China
c
Center for Environmental and Genetic Medicine, Texas A&M Health Science Center, Institute of Biosciences and Technology, Houston, Texas, USA

Emissions of complex mixtures of polycyclic aromatic hydrocarbons (PAHs) and other compounds into the environment represent a potential threat to
the health of humans. Information regarding the dose and duration of exposure is essential to determine the degree of risk and to identify sensitive
receptors within a population. Although measurements of chemical concentrations in air may be used to estimate exposures, internal biomarkers provide
more accurate information regarding the dose of exposure and retention of toxic chemicals. This study was conducted in a population in rural China
exposed to PAHs from a variety of sources. The study population was located in an area known to have an elevated incidence of birth defects. Parents of
children born with a neural tube defect (NTD) were recruited as case participants and parents of children born with no visible birth defect were recruited
as controls. The study was designed to test the hypothesis that parents of children born with a NTD would exhibit a biomarker of exposure at higher
levels than the parents of a child with no visible birth defect. A total of 35 mothers and 32 fathers were recruited as case participants, and 18 mothers and
19 fathers were recruited as control participants. Venous blood was collected from the study participants by hospital staff as soon as possible following the
birth of the child. PAHs were isolated from the whole blood by solvent extraction and DNA was isolated from a separate aliquot of blood for 32P-
postlabeling to measure bulky adducts. Single Nucleotide Polymorphisms (SNPs) in phase II enzymes were also monitored in an attempt to identify
sensitive receptors. Both total and carcinogenic PAH (cPAH) concentrations were elevated in the parents of case children. Both values were elevated
significantly in mothers, whereas only cPAH concentrations were elevated significantly in fathers. Levels of DNA adducts were highly variable and
displayed a reverse pattern to that of PAH levels in blood. None of the polymorphisms evaluated were correlated with PAH levels or DNA adducts. For
mothers, whose total PAH concentration was above the median concentration, the age-adjusted odds ratio (OR) for having a child with a NTD was 8.7.
Although this suggests that PAHs may be a contributing factor to the risk of NTDs, the lack of a correlation with DNA adducts would suggest a possible
non-genotoxic mechanism. Alternatively, the PAHs may be a surrogate for a different exposure that is more directly related to the birth defects. The
results have shown that blood levels of PAHs may be used to identify populations exposed to elevated concentrations of combustion by-products.
Journal of Exposure Science and Environmental Epidemiology (2010) 20, 310–319; doi:10.1038/jes.2009.19; published online 11 March 2009

Keywords: PAHs, biomarkers, China, human blood.

Introduction organic substances, such as coal, gas, wood, or tobacco, are

Humans are constantly exposed to hazardous chemicals.
These chemicals are usually mixtures and may contain
hundreds of different components. Often, complex mixtures
include components that are known to be toxic to human or
ecological receptors and others with a less well-characterized
toxicity. Polycyclic aromatic hydrocarbons (PAHs), a class
of chemicals produced from the incomplete combustion of
1. Address all correspondence to: Dr Kirby C Donnelly, Department of
Environmental and Occupational Health, Texas A&M Health Science
Center, School of Rural Public Health, 1266 TAMU, College Station, TX
77843-1266, USA. Tel.: þ 1 979 845 7956. Fax: þ 1 979 845 0885.
E-mail: kdonnelly@cvm.tamu.edu or
Guo-dong Zhou. Tel.: þ 1 713 677 7433. Fax: þ 1 713 677 7689.
E-mail: gzhou@ibt.tamhsc.edu
Received 20 October 2008; accepted 26 January 2009; published online
11 March 2009
one of the most common complex mixtures in the environ-
ment. Several PAHs have been classified as carcinogens by
the US. Environmental Protection Agency (USEPA) (USE-
PA, 2006) and the International Agency for Research on
Cancer (IARC, 2004). Epidemiological studies on popula-
tions exposed to PAH mixtures have shown a link between
these exposures, and cancers of the lung, respiratory system,
and stomach (Bertrand et al., 1987; Puisieux et al., 1991;
Krewski and Thomas, 1992; Vyskocil et al., 2004). Smoking
and exposure to environmental tobacco smoke (ETS) have
been shown to be associated with cancer in the lung,
bronchus, larynx, bladder, cervix, and oral mucosa (Phillips,
1997; Lee et al., 2006; Yach and Wipfli, 2006).
PAH exposure levels are classically estimated by air
sampling. Air sampling provides valuable information
regarding environmental concentrations and potential human
exposures. However, air sampling does not provide information
Biomarkers of PAHs in human blood
regarding internal dose. Internal dose may be quantified by
the determination of a parent compound or metabolite in
body fluids such as blood or urine. Biological effects, as a
measure of internally effective dose, are considered more
relevant for the assessment of the ultimate human health risk.
These effects are monitored by biochemical markers,
including covalent binding products to DNA or proteins in
addition to DNA strand breaks, or cytogenetic markers, such
as micronuclei, chromosome aberrations, and sister chroma-
tid exchange (van Delft et al., 2001). DNA adducts are
considered as a marker for potential risk of genotoxic effects,
which might lead to undesirable outcomes, such as cancer or
birth defects. DNA adducts may also reflect individual
variations in exposure, absorption, metabolic activation, and
DNA repair. The estimated half-life of DNA adducts was
reported to be approximately 3–4 months (Mooney et al.,
1995). Hemminki et al. (1990a, b) monitored DNA adducts
and PAH exposures in coke oven workers in Poland. Two
control groups were recruited to the study. One group
consisted of local residents who lived in the area, but were not
coke oven workers. The second control group consisted of
rurally residing non-coke oven workers. Unexpectedly, DNA
adduct concentrations in the local controls were nearly as
high as the levels detected in the coke oven workers.
However, the adduct levels in the rural controls were two
to three times lower than those of the coke oven workers, as
expected. The authors suggested that DNA adduct levels in
those non-coke oven workers who lived in the surrounding
area were similar to the concentrations measured in the coke
oven workers as a result of environmental pollution in the
area. The authors noted that this locality is a highly
industrialized region, with a large population and the
presence of many coal mines, smelters, and chemical plants.
The authors also noted that the common practice of domestic
coal burning may have also been a source of exposure for the
local residents. DNA adducts measured by 32P-postlabeling
in WBCs of a non-smoking group of women who work
outdoors in a polluted city in the Czech Republic was
associated with their personal PAH exposure (Binkova et al.,
1995). In a more recent study, Peluso et al. (2008) studied the
genotoxic effects of air pollutant exposures on a human
population living downwind to an industrial complex in
Thailand. Their findings revealed that the level of bulky
DNA adducts in the leukocytes of residents was 0.85±0.07
per 108 nucleotides, which was significantly elevated com-
pared with the residents living in a control district.
Many studies on environmental exposures to PAHs in
humans focus on air pollution. Epidemiological studies
indicate that exposures to PAHs in air are associated with
increases in mortality and/or morbidity from respiratory
illnesses, cardiovascular diseases, cancer (Taioli et al., 2007),
and adverse birth outcomes. Nielsen et al. (1996) observed a
relative risk for lung cancer of 1.5 in an urban community
exposed to air pollution. Airborne PAHs have been
Journal of Exposure Science and Environmental Epidemiology (2010) 20(4)
Naufal et al.
implicated in human reproductive effects, notably DNA
adducts and hypoxanthine-guanine phosphoribosyltransfer-
ase mutations in newborns, as well as preterm birth and
intrauterine growth restriction (Srám et al., 1999; Dejmek
et al. 2000; Perera et al. 2002). Findings from a more recent
study suggest that high levels of PAHs, as measured by DNA
adducts in cord blood from the World Trade Center fires in
New York City in 2001, may have contributed to reduced
fetal growth in exposed women (Perera et al. 2005). In a
review of the literature on ambient pollution and pregnancy
outcomes by Srám et al. (1999), low birth weight, premature
birth, and intrauterine growth retardation seemed to be the
reproductive effects most often associated with air pollutants.
The study observed that intrauterine growth retardation was
specifically linked with PAHs. A potential link between coal
combustion by-products and the risk of congenital
malformation was investigated by a study conducted in
Nova Scotia, Canada. In this study, the risk of congenital
malformations (overall and specific categories) was increased
in the offspring of the residents of a coking operation,
compared with the rest of Nova Scotia (Dodds and Seviour,
2001). On the other hand, a recent report (Shaw et al. 2009)
suggests that maternal smoking, measured with cotinine
levels during mid-pregnancy, was possibly associated with
reduced risks of neural tube defects (NTDs).
Single nucleotide polymorphisms (SNPs) that alter the
activity of metabolizing or DNA repair enzymes are also
assumed to alter the formation of DNA adducts. Several
studies have noted a lack of significant difference in
lymphocyte PAH-DNA adduct levels between GSTM1-
deficient and -proficient individuals (Ryberg et al., 1994;
Hou et al., 1995; Binkova et al., 1998). However, findings
reported by Georgiadis et al. (2004) indicate that non-
smoking students exposed to urban air pollution and ETS
with GSTM1 deletion had higher levels of DNA adducts in
their lymphocytes compared with GSTM1 ‘‘wild-type’’
participants. Never-smoking women with GSTM1 null had
statistically significant greater risk of developing lung cancer
from exposure to ETS (Bennett et al., 1999). Lung cancer
risk in GSTM1-null individuals exposed to indoor coal
combustion emissions was elevated compared with indivi-
duals with an active copy of GSTM1. GSTT1 polymorph-
isms, on the other hand, did not seem to be associated with
lung cancer risk (Lan et al., 2000). However, individuals with
null genotypes of GSTT1 seemingly had an increased risk for
developing colorectal cancers (Deakin et al., 1996). The
inactive GSTT1 genotype was also associated with increased
risk in some forms of brain tumors (Hand et al., 1996; Kelsey
et al., 1997).
In an earlier report, elevated levels of genotoxic mixtures of
PAHs were detected in the residential environments of a
population located in Shanxi province, China (Naufal et al.,
2007). The study described in this paper was directed at the
analysis of biomakers of exposure, effect, and susceptibility
311
 Naufal et al.
in parents of children born with NTDs, and their matched
controls. As a measure of internal dose of PAHs, PAH
concentrations in whole blood were quantified. The fre-
quency of aromatic DNA adducts was measured by 32P-
postlabeling technique in WBCs from recruited participants
as an indicator of internal effective dose. Polymorphisms of
two main phase II metabolic enzymes (GSTM1, GSTT1)
were also monitored to evaluate the influence of genetic
sensitivities.
Methods
Study Site
Four counties (Taigu, Pingding, Xiyang, and Zezhou) in
Shanxi province in northern China were selected for this
research as it was anticipated that elevated concentrations of
PAHs were present in their environment. Shanxi is located
approximately 200 miles Northwest of Beijing. Shan-
xiFknown as the ‘‘Coal Warehouse of China’’Fis the
leading province in coal production in China and provides as
much as one quarter of China’s coal. The coal mined from
this region is most often used indoors for cooking and
heating. Residents in Shanxi may be continuously exposed to
PAHs and other by-products by either inhalation of the
airborne soot particles or ingestion of soot particles that
deposit on food. The incidence of NTDs in Shanxi is one of
the highest in the world. There is concern regarding the
impact of PAH exposures on health because the overall
prevalence rate for NTDs in China is 1.3/1000 live births;
whereas in the Northern part of the country (where this study
was conducted), the rate is 2/1000 births (Dai et al., 2002).
In comparison, the estimated NTD prevalence in the United
States, based on birth certificate data for 1995, was 0.4/1000
(Mathews et al., 2002).
Participant Recruitment
This study focused on parents of children with congenital
abnormalities because it was believed that these may
represent a segment of the population sensitive to PAH
exposures. Study participants included parents of
children born with a NTD and parents of children born
with no visible abnormality at the same hospital and
conceived at approximately the same time to serve as a
control group. A control was recruited as the first baby to be
born with no visible malformation within 48 h of a case
recruitment in the same hospital. Participants were recruited
for participation in the study throughout the year at four
county hospitals in Taigu, Pingding, Xiyang, and Zezhou.
The combined annual number of births at these four
hospitals was on average more than 3000; prevalence rates
for NTDs in the four hospitals ranged from approximately
8/1000 to 24/1000. Overall, NTDs did not seem to exhibit
seasonal variations.
312
Biomarkers of PAHs in human blood
The types of congenital malformations that were studied
were selected on the basis of evidence suggesting an
environmental component in their etiology. Neural tube
defects, including anencephaly and spina bifida, are readily
recognizable at birth based on a routine newborn physical
examination. Participant recruitment was facilitated by the
presence of a birth defects surveillance system in China
since 1992 as described by Li et al. (2003). All recruited
participants were informed of the nature of the study, and
signed consent forms approved by the Texas A&M
University Institutional Review Board (IRB no. 2003-
0430). Mothers who provided informed consent to enroll in
the study were interviewed in person using a standardized
questionnaire that was translated to Chinese. The ques-
tionnaire was designed to obtain information relevant to
potential risk factors and confounders that are relevant to the
circumstances in China.
Sample Collection
Venous Blood Samples Venous blood samples were
collected at the hospital by the medical staff and stored at
41C before transporting them to the Peking University
Health Science Center in Beijing, China. A volume of 10–
15 ml of venous blood was collected in sodium heparin BD
Vacutainer tubes (VWR, catalogue no.VT6480) from study
participants. Samples were stored at appropriate
temperatures to be shipped on refrigerant gel packs to
Texas A&M University for processing and analysis.
Shipment of samples was carried out according to the
regulations of the US Department of Transportation and
International Air Transport Association.
Sample Extraction and Chemical Analysis
Extraction of Venous Blood Venous blood samples were
centrifuged in the heparinized collection tubes at 3000 RPM
for 10 min. This procedure served to separate the blood into
an upper lighter plasma layer and a lower denser cell layer.
The plasma layer was transferred by pipette to borosilicate
glass vials to store at 801C for PAH extraction and
analysis. The cell layer was stored in the original blood
collection tubes at 41C for DNA isolation.
A modified version of the method described in the USEPA
Manual of Analytical Methods for the Analysis of Pesticides
in Human and Environmental Samples (Watts, 1980) was
used to extract PAHs from the plasma using a liquid–liquid
extraction. A volume of 2 ml of plasma was used. Surrogates
consisting of deuterated PAHs, such as naphthalene,
acenaphthylene, phenanthrene, chrysene, and perylene, were
added before sample extraction. The plasma was thawed and
subsequently mixed with methanol (1:2 v/v) to denature
plasma proteins. Samples were then sonicated thrice for 1
minute at approximately 5-minute intervals. A break during
Journal of Exposure Science and Environmental Epidemiology (2010) 20(4)
Biomarkers of PAHs in human blood
sonication was needed to prevent heating of the samples.
Samples were then decanted into separatory funnels, and
10 ml of dichloromethane was added to begin the extraction
process and to rinse each sample vial. Plasma samples were
then extracted thrice with 20 ml of dichloromethane. Each
extract was shaken for 3 min with a 20-min settling period.
The combined extracts were filtered on a solid phase
extraction column (Restek, Bellefonte, PA, USA) that
contained a layer of combusted sodium sulfate. The volume
of the extract was reduced to 2–3 ml before chemical analysis.
Chemical Analysis A modified USEPA SW-846 Method
8270C (USEPA, 1996) was used for the quantitative
determination of PAHs and their alkylated homologs in
extracts of biological tissue. This method was developed for
PAH quantitation and was described earlier (Cizmas et al.,
2003). Analysis was conducted on a Hewlett-Packard 5890
Series II gas chromatograph with a 5972 mass selective
detector in selected ion monitoring mode. A 60 m  0.25 mm
ID  0.25 mm film thickness DB-5MS column (Agilent
Technologies, Palo Alto, CA, USA) was used. The
injection port was maintained at 3001C and the transfer
line at 2801C. The temperature program was as follows:
601C for 6 min increased at 121C/min to 1801C, then
increased at 61C/min to 3101C and held for 11 min for a total
run time of 47 min. The method limit of detection (MDL) for
benzo[a]pyrene (B(a)P) in blood was estimated at 2.95 ng/ml.
Recovery of surrogates was between 40% and 110%.
DNA Isolation and 32P-Postlabeling
DNA isolation from WBCs was conducted as described
earlier (Gupta, 1984; Binkova et al., 2000). DNA concen-
tration and purity were measured spectrophotometrically by
absorbances at 260 and 280 nm. The A260/A280 ratio for all
samples was between 1.6 and 1.8.
The nuclease P1-enhanced bisphosphate version of the
32
P-postlabeling assay was performed as described by
Reddy and Randerath (1986). Briefly, 10 mg of DNA was
enzymatically digested to normal (Np) and modified (Xp)
deoxyribonucleoside 30 -monophosphates. After 30 -depho-
sphorylation of normal nucleotides with nuclease P1, the
enriched nuclease P1-resistant modified 30 -nucleotides were
converted to 50 -32P-labeled deoxyribonucleoside 30 and
50 -bisphosphate derivatives (pXp) by incubation with car-
rier-free [g-32P] ATP and T4 polynucleotide kinase. Ad-
ducted radioactive nucleotides were separated by
multidirectional anion-exchange thin-layer chromatography
(TLC) using polyethyleneimine-cellulose sheets. Labeled
products were purified and partially resolved by one-
dimensional development with 2.3 M NaH2PO4, pH 5.75
overnight (D1). Bulky labeled adducts retained in the lower
(2.8  1.0 cm) part of the D1 chromatogram were contact-
transferred to fresh thin-layer sheets and resolved by two-
dimensional TLC. The first dimension employed 3.82 M
Journal of Exposure Science and Environmental Epidemiology (2010) 20(4)
Naufal et al.
lithium formate þ 6.75 M urea, pH 3.35. The second dimen-
sion was developed with 0.72 M NaH2PO4 þ 0.45 M
TRIS þ 7.65 M urea, pH 8.2. Radioactivity of each TLC
map was determined by using an Instant Imager (Packard
Instrument, Downers Grove, IL, USA). DNA adduct levels
were quantified as mean relative adduct labeling (RAL)
values±SD using the following equation:
RAL ¼ sample count rate=ðDNA Pspecific activityATP Þ
where the sample count rate is measured in cpm, DNA-P
represents the pmol of DNA monomer units assayed per
replicate, and the specific activity of the ATP is in units of
cpm/pmol.
Genotyping
DNA from WBC samples was genotyped for null deletions in
GSTM1 and GSTT1 using the methods described by Bailey
et al. (1998). PCR was used to amplify and assay the
GSTM1 and GSTT1 alleles. The products were digested with
the restriction enzymes HinfI and NcoI, electrophoresed, and
visualized using ethidium bromide staining. The genotyping
call rate was about 99.6%.
Statistical Analyses
Statistical analyses were performed using SigmaStat (version
3.1) software package. Multivariate analyses were performed by
using Student’s t-test or one-way Analysis of Variance
(ANOVA) unless the normality test failed (Po0.05). When
normality failed, Mann–Whitney Rank Sum Test or Kruskal–
Wallis one-way ANOVA on Ranks was carried out. Pearson’s
coefficient was computed to assess bivariate correlations. The
criterion for statistical significance was set at Po0.05. w2and
Fisher’s exact tests were used to compute a crude odds ratio
(OR) and 95% confidence interval (CI). The age-adjusted OR
was calculated using logistic regression, including maternal age
as a covariate in the model, with the outcome being a NTD case
versus a normal birth as a control.
Results
Venous blood was collected from mothers and fathers of
children born with a NTD, and parents of a matched control
child. Samples were collected from a total of 53 mothers and
51 fathers. Among the mothers, 35 delivered a baby with a
NTD and 18 delivered visibly normal babies, and were
recruited as controls. Among the fathers, 32 were the parents
of a baby with a NTD and 19 were fathers of visibly normal
children. None of the mothers reported to be a smoker.
However, 27 (18 cases, 9 controls) of 53 mothers reported
being exposed to passive cigarette smoke. The majority of the
mothers were aged between 25 and 35 years. Among the
fathers, 38 (25 cases, 13 controls) of 51 reported smoking, at
313
 Naufal et al. Biomarkers of PAHs in human blood

least occasionally. The characteristics of the study population
are summarized in Table 1.
As a measure of internal dose, extracts of venous blood
samples from mothers and fathers were chemically char-
acterized for 60 PAH congeners. The chemicals quantified in
blood included low and high molecular weight PAHs, in
addition to their alkylated homologs. The seven PAHs
classified by the USEPA as class B2 carcinogens were also
included in the analysis. The mean level of total PAHs
(tPAHs) detected in venous blood from mothers of cases
(258 ng/ml) was significantly higher than that detected in
venous blood from control mothers (120 ng/ml) (Table 2).
Venous blood from case fathers was found to have mean
PAH levels of 217 ng/ml, whereas mean PAH levels of
159 ng/ml were detected in blood from control fathers.
However, this difference was not statistically significant.
The seven USEPA class B2 carcinogenic PAHs (cPAHs)F
benz(a)anthracene, chrysene, benzo(b)fluoranthene, ben-
zo(k)fluoranthene, B(a)P, indeno(1,2,3-c,d)pyrene, and
dibenzo(a,h)anthraceneFwere also quantified in venous blood
samples from parents of case and control children. Mean levels
of cPAHs were significantly elevated in case mothers (19 ng/ml)
when compared with control mothers (9 ng/ml). Among
fathers, those with case children had mean cPAH levels of
15 ng/ml, whereas the concentration of cPAHs in venous blood
in case fathers were also statistically elevated in comparison with
control fathers. The data on cPAHs among case and control
mothers, as well as case and control fathers are presented in
Figure 1.
Although none of the mothers smoked, 27 mothers
reported being exposed to passive cigarette smoke. Mothers
exposed to passive smoking (occasional or daily) had mean
levels of tPAHs and cPAHs in their blood of 233 and 16 ng/
ml, respectively. Non-smoking mothers who reported no
exposure to passive smoking had mean levels of 188 ng/ml
tPAHs and 15 ng/ml cPAHs. However, these differences
were not statistically significant.
Smoking among fathers was very common (38/51) and
included a range of participants from occasional smokers to
individuals who smoked more than 20 cigarettes per day.
Fathers were separated into two subsets by smoking status to
detect any difference in the tPAH and cPAH levels among
smokers and non-smokers. Non-smoking fathers had mean
tPAH levels of 236 ng/ml in their venous blood compared
with 182 ng/ml for smokers, but the difference was not
statistically significant.
25
Case
Mean Concentration (ng/mL)

*
from fathers of control children was 10 ng/ml. The cPAH levels Control
20
*
Table 1. Characteristics of the adult population recruited for this 15
study.
10
Case N (%) Control N (%) Total

Mothers 35 (66) 18 (34) 53 5

Age at delivery (years)
o25 12 (71) 5 (29) 17 0
25–35 17 (59) 11 (41) 28 Mother Father
435 5 (71) 2 (29) 7 N=35(Case); 18(Control) N=32(Case); 19(Control)
Exposed to passive smoking 18 (67) 9 (33) 27 *P < 0.05

Fathers 32 (63) 19 (37) 51 Figure 1. Concentrations of carcinogenic PAHs detected in venous

Smoker 25 (66) 13 (34) 38 blood collected from parents of children born with neural tube defects
(case) and parents of control children.

Table 2. Total and carcinogenic PAHs detected in venous blood from mothers and fathers (ng/ml).

tPAHs cPAHs

N Min Max Mean±SEM Min Max Mean±SEM

Mothers
Case 35 29 762 258±26 Po0.01 0.2 65 19±3 Po0.05
Control 18 33 234 120±16 1 49 9±3

Fathers
Case 32 52 523 217±27 P ¼ 0.25 1 50 15±2 Po0.05
Control 19 25 345 159±23 1 48 10±3

314 Journal of Exposure Science and Environmental Epidemiology (2010) 20(4)

Biomarkers of PAHs in human blood Naufal et al.

The exposures in mothers and fathers were further as a cooking fuel were compared with those who use another
evaluated. A linear regression was analyzed to investigate type of cooking fuel, they seemed significantly elevated. In
the possible correlation between tPAH levels in blood from fact, mean levels of DNA adducts in WBCs of mothers using
paired fathers and mothers (Figure 2a). A correlation that hard coal were 15 per 109 nucleotides which was significantly
was statistically significant was detected (correlation coeffi- elevated compared with 11 adducts per 109 nucleotides, the
cient (r) ¼ 0.408; Po0.05). In addition, a linear regression of mean level found in WBCs of mothers using other types of
the levels of blood tPAHs in paired non-smoking fathers and
mothers revealed a stronger statistically significant correlation Table 3. Estimated risk of NTDs based on concentration of PAHs
(r ¼ 0.662; Po0.05) (Figure 2b). measured in whole blood extracts from mothers.
The association between having a baby with a NTD and
maternal PAH exposure level was examined. Mothers were Exposure Cases Controls Crude OR Age-adjusted
divided into two groups, a high exposure group and a low levela (95% CI) OR (95% CI)
exposure group, according to their median blood PAH level
High 24 3 10.91 (2.61–45.60) 8.70 (2.02–37.04)
(206 ng/ml). Mothers with high PAH exposures were found Low 11 15
to have a significantly higher risk compared with the lower Total 35 18
exposure group (OR 10.91; 95% CI: 2.61–45.60). The age-
Abbreviations: CI, confidence interval; OR, odds ratio.
adjusted model did not change the increased risk of NTDs a
High exposure was defined as a blood total PAH level greater than the
among mothers (OR 8.70; 95% CI: 2.02–37.04) (Table 3). median (206 ng/ml); low was defined as a blood PAH level lower than the
Bulky DNA adducts were analyzed by 32P-postlabeling in median.
WBCs as a biomarker of genetic damage. Control parents
exhibited significantly higher levels of DNA adducts as
compared with case parents. Figure 3 displays bar graphs 20
*
Mean RAL per 109 nucleotides

representing mean DNA adduct levels detected in WBCs of 18

mothers. Mean level of DNA adducts in case mothers was 16
12.19 per 109 normal nucleotides compared with 17.13 per 14
109 nucleotides in controls. Similarly, mean level of DNA 12
adducts in case fathers was 10.97 per 109 nucleotides, which 10
was significantly lower than 20.64 per 109 nucleotides, the 8
mean level of bulky DNA adducts in control fathers. 6
Smoking status did not seem to influence the levels of 4
DNA adducts in the study participants. 2
The variable that seemed to have the greatest impact on 0
Case Control
DNA adduct levels in mothers was the type of fuel used for
N=35 N=18
cooking. Mothers reported using types of hard coal, black Mothers *P<0.05
coal, firewood, and natural gas as cooking fuels. Hard coal 9
Figure 3. Frequency of bulky DNA adducts (per 10 nucleotides)
seems to be the most abundantly used cooking fuel. When detected in venous blood from mothers of children born with neural
DNA adduct levels in WBCs of mothers who used hard coal tube defects (case) and mothers of control children.
Total PAHs in Venous Blood of

600 500
Non-Smoking Fathers
Total PAHs in Venous

500 400
Blood of Fathers

400
300
300
200
200
N=37 N=12
100 100
r=0.408 r=0.662
P<0.05 P<0.05
0 0
0 200 400 600 800 0 50 100 150 200 250 300
Total PAHs in Venous Total PAHs in Venous Blood of
Blood of Mothers Mothers with Non-Smoking Husbands
Figure 2. Linear regression showing the correlation between total PAHs in venous blood from fathers and total PAHs in venous blood from mothers
(a), and that between total PAHs in venous blood restricted to non-smoking fathers and total PAHs in venous blood restricted to mothers with non-
smoking husbands (b), and the corresponding P-values. Units are in ng/ml.

Journal of Exposure Science and Environmental Epidemiology (2010) 20(4) 315

 Naufal et al. Biomarkers of PAHs in human blood

18 Table 4. Ratio of bulky DNA adducts to carcinogenic PAHs in

Mean RAL per 109 nucleotides

16 * venous blood based on GSTM1 and GSTT1 polymorphisms

14
Exposure levela GSTT1 GSTM1
12
10 W/t Null W/t Null
8
High
6
Mean 0.70 0.63 0.62 0.70
4 SEM 0.09 0.14 0.11 0.12
2 N 25 15 23 18
0 P-value 0.663 0.631
Hard Coal Other
N=34 N=14 Low
Cooking Fuel *P < 0.05 Mean 5.20 4.61 4.02 6.07
SEM 0.76 1.04 0.61 1.10
Figure 4. Frequency of bulky DNA adducts in venous blood collected N 26 12 22 17
from mothers by the type of fuel used for cooking. P-value 0.656 0.094

cooking fuel. These data are graphically represented in
Figure 4.
Genotyping of two major phase II metabolic enzymes,
GSTM1 and GSTT1, revealed that the GSTM1 deletion rate
was of 26% in mothers and 19% in fathers, whereas GSTT1-
null genotype was found in 19% of the mothers and 19% of
the fathers. The effect of having an inactive copy of GSTM1
or GSTT1 was evaluated by computing ratios of PAH levels
in blood to levels of bulky DNA adducts in study
participants, combining data from fathers and mothers.
The assumption was that an inactive form of GSTM1 or
GSTT1 might slow down the elimination of PAH com-
pounds and thereby increases the likelihood of DNA adduct
formation by these chemicals. Thus, the ratio of DNA
adduct to PAH blood level in individuals with non-functional
GSTM1 or GSTT1 was expected to be higher than in
individuals with a functional copy of these enzymes. Further,
individuals were grouped by exposure level as determined by
their cPAH blood levels. High exposure level was defined as
cPAH higher than the median, whereas lower exposure level
was defined as a cPAH level lower than the median. In the
low exposure group, the mean ratio detected in GSTM1-null
individuals (6.07) was higher than that in GSTM1 wild-type
individuals (4.02), achieving marginal statistical significance
(Po0.1). In the high exposure group, the same trend was
observed, however, without statistical significance. The
results were reversed across GSTT1 polymorphisms, without
being statistically significant. These data are presented in
Table 4.
Discussion
The goal of this study was to collect biological samples to
evaluate two different methods for measurements of bio-
markers in a population exposed to combustion by-products.
Data were collected to test the hypothesis that the mothers or
a
High exposure was defined as a blood carcinogenic PAH level greater than
the median (8 ng/ml); low was defined as a blood PAH level lower than the
median.
fathers of children born with a NTD would exhibit a
biomarker of exposure at higher levels than the parents of a
child with no visible birth defect. A total of 53 mothers (35
cases and 18 controls) and 51 fathers (32 cases and 19
controls) were recruited to participate in the study. Earlier
studies have observed elevated rates of birth defects in the
provinces where the participants were recruited (Li et al.,
2006; Gu et al., 2007). It was also anticipated that the
population has multiple sources of exposure to PAHs,
including ingestion of cooked foods, inhalation of coal
combustion by-products, as well as active and passive
smoking (in approximately half the population). Any
chemical at a certain dose level can arguably induce
teratogenic effects. During early pregnancy, when organs
are developing, the fetus is especially vulnerable. Maternal
chemical exposures can, therefore, significantly affect fetal
development by compromising placental development and
exchange, as well as hormonal signaling needed for in utero
fetal growth (Miller et al. 2004). PAHs can affect in utero
fetal development by binding to the aryl hydrocarbon
receptor (AhR) causing anti-estrogenic activity, through
increased metabolism and depletion of endogenous estrogens,
(Carpenter et al. 2002) thus disrupting the endocrine system
by altering steroid function. In addition, PAH binding to
receptors for placental growth factors may result in an
impairment of the feto-placental oxygen and nutrient
exchange (Dejmek et al. 2000) leading to fetotoxicity.
The data were collected from case and control parents for
two biomarkers. Concentrations of total and carcinogenic
PAHs were used to provide information on total exposures to
combustion by-products. Measurement of DNA adducts
were used to provide a biomarker of genotoxic effects. The
incidence of DNA adducts is believed to be affected both by

316 Journal of Exposure Science and Environmental Epidemiology (2010) 20(4)

Biomarkers of PAHs in human blood
total exposure, as well as genetic factors that influence
retention of PAHs and repair of DNA adducts. The results
confirmed significant differences in biomarkers between case
and control participants. Specifically, both total and cPAHs
were significantly higher in blood from case mothers when
compared with control mothers. For fathers, 75% of whom
report smoking, there was no significant difference in tPAH
levels between case and control participants. However,
cPAHs were significantly higher in the case fathers. The
correlation between the levels of tPAHs in the blood of
paired fathers and mothers suggests the presence of a
common source of exposure. When adjusted for smoking,
the correlation became more pronounced indicating further
that the source of PAHs is primarily environmental. Indoor
coal combustion seems to be the chief source of PAH
exposure in the study population.
Results from other studies, which investigated the levels of
PAHs in blood for comparison purposes, are limited. Singh
et al. (2008) evaluated nine individual PAHs in the blood of
an Indian population. In addition, Neal et al. (2008)
evaluated four specific PAHs in a Canadian population.
This work presented a total of approximately 60 analyzed
PAH congeners (low- and high-molecular weight PAHs, and
alkylated homologs). As the PAH data are presented
differently, direct comparison of quantified values is not
possible. However, results from a similar biomarker study
currently evaluating a similar panel of PAHs in a population
living near a hazardous waste site in Sumgayit, Azerbaijan,
indicate that the levels of blood PAHs in study participants
are similar to those found in our study participants in China
(unpublished data).
Bulky DNA adducts exhibited a reversed trend and were
significantly higher in both parents of controls as compared
with NTD cases. This trend suggests a potential non-
genotoxic mechanism of PAHs in the etiology of NTDs. An
earlier report indicated that PAHs are potentially capable of
inducing estrogenic and anti-estrogenic responses in humans
mediated by the activation of the estrogen receptor (ER)
(Santodonato, 1997). Recent studies provided further
experimental evidence that PAHs, more specifically B(a)P
and 3-methylcholanthrene, in addition to being AhR
agonists can also bind and activate ERa independently of
AhR (Abdelrahim et al., 2006; Liu et al., 2006). Organic
extracts primarily consisting of polycyclic aromatic com-
pounds, isolated from road dust and diesel exhaust
particulates, were found to contain contaminants that
induced significant ER ligand activity (Misaki et al., 2008).
Nevertheless, further research is needed to better understand
these relatively less documented mechanisms of PAH toxicity
and their effects on living organisms. Alternatively, the PAHs
may serve as surrogate markers for exposures to other
components of combustion by-products (e.g., arsenic) and
thus a genotoxic biomarker was not induced in the parents of
children with a birth defect. Arsenic has been linked with
Journal of Exposure Science and Environmental Epidemiology (2010) 20(4)
Naufal et al.
NTDs in animal studies (ATSDR, 2007). However, limited
measurements of arsenic in coal and drinking water in this
region have not indicated elevated concentrations of arsenic
(unpublished data).
Other explanations for the lack of correlation between
PAH levels in blood and DNA adducts include the fact that
DNA adducts were analyzed in total leukocytes as opposed
to specific long-lived WBCs, which might lead to a dilution
effect in DNA adduct levels. Route of exposure may also
affect DNA adduct concentrations detected in WBCs.
Specifically, adduct levels following inhalation exposure
(the main route of exposure in this study) may be lower
than levels detected following oral or dermal exposure. In
addition, the non-cPAHs may have altered metabolism of
the cPAHs and thus reduced the formation of DNA adducts.
Studies of complex PAH mixtures in animals suggest that this
type of inhibitory effect of PAH mixtures is common
(ATSDR, 1995).
Cooking using hard coal seemed to have the greatest
influence on increasing the levels of DNA adducts in
mothers. The use of solid fuel for cooking was associated
earlier with an increased risk of lung cancer in Europe
(Lissowska et al., 2005). In China, cooking frequency was
associated with higher levels of urinary 1-hydroxypyrene in
females as well as males (Chen et al., 2007).
Deletions in two major phase II metabolic enzymes
GSTM1 and GSTT1 were evaluated as a measure of genetic
sensitivity in this population. Individuals with inactive copies
of GSTM1 exhibited higher ratios of DNA adducts to cPAH
blood levels compared with those with active forms of these
enzymes. Evaluation of genetic variations in additional genes
is warranted for better understanding of the genetic
sensitivities present in the study population.
In conclusion, the presence of genotoxic PAH compounds
was confirmed in biological tissues sampled from a human
population located in a rural area of China. One limitation of
this investigation is the sample size and the limited number of
controls per case. Results from this work are preliminary and
should be confirmed in a larger-scale study. Mothers of
newborn children with a NTD exhibited elevated levels of
PAHs in their blood as compared with controls. A relation-
ship between the ratios of DNA adducts to exposure and
single nucleotide polymorphisms in phase II enzymes was
observed. Although data were not obtained to identify a
mechanism to associate the PAH exposures with the birth
defects, the results suggest that it is seemingly not associated
with a genotoxic response as measured by DNA adducts.
Acknowledgements
We thank the staff at all the participating county hospitals
in Shanxi, China. We also wish to acknowledge the
US Environmental Protection Agency (USEPA) and the
317
 Naufal et al.
National Institute of Environmental Health Sciences Super-
fund Basic Research Program (NIEHS SBRP) for sponsor-
ing this work. Kristin Marano is recognized for her excellent
editorial support.
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