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. DONNELLYa

aDepartment of Environmental and Occupational Health, Texas A&M Health Science Center, School of Rural Public Health, College Station, Texas, USAbInstitute of Reproductive and Child Health, Peking University Health Science Center, Beijing, ChinacCenter 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

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

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

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 informationReceived 20 October 2008; accepted 26 January 2009; published online

11 March 2009

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

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

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

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 (Sram 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 Sram 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

Biomarkers of PAHs in human blood Naufal et al.

Journal of Exposure Science and Environmental Epidemiology (2010) 20(4) 311

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 SiteFour 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 RecruitmentThis 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.

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–

15ml 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 10min. 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 2ml 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

Biomarkers of PAHs in human bloodNaufal et al.

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

sonication was needed to prevent heating of the samples.

Samples were then decanted into separatory funnels, and

10ml of dichloromethane was added to begin the extraction

process and to rinse each sample vial. Plasma samples were

then extracted thrice with 20ml of dichloromethane. Each

extract was shaken for 3min 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 2–3ml 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 60m� 0.25mm

ID� 0.25mm 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 6min increased at 121C/min to 1801C, then

increased at 61C/min to 3101C and held for 11min for a total

run time of 47min. 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-PostlabelingDNA 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 the32P-postlabeling assay was performed as described by

Reddy and Randerath (1986). Briefly, 10mg 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 2.3M 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 3.82M

lithium formateþ 6.75M urea, pH 3.35. The second dimen-

sion was developed with 0.72M NaH2PO4þ 0.45M

TRISþ 7.65M 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�P�specific activityATPÞ

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.

GenotypingDNA fromWBC 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 AnalysesStatistical 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. w2andFisher’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

Biomarkers of PAHs in human blood Naufal et al.

Journal of Exposure Science and Environmental Epidemiology (2010) 20(4) 313

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)Fbenz(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 (19ng/ml)

when compared with control mothers (9ng/ml). Among

fathers, those with case children had mean cPAH levels of

15ng/ml, whereas the concentration of cPAHs in venous blood

from fathers of control children was 10ng/ml. The cPAH levels

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.

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 P¼ 0.25 1 50 15±2 Po0.05

Control 19 25 345 159±23 1 48 10±3

0

5

10

15

20

25

MotherN=35(Case); 18(Control)

Mea

n C

on

cen

trat

ion

(n

g/m

L)

*P < 0.05

*

*

CaseControl

FatherN=32(Case); 19(Control)

Figure 1. Concentrations of carcinogenic PAHs detected in venousblood collected from parents of children born with neural tube defects(case) and parents of control children.

Table 1. Characteristics of the adult population recruited for thisstudy.

Case N (%) Control N (%) Total

Mothers 35 (66) 18 (34) 53

Age at delivery (years)

o25 12 (71) 5 (29) 17

25–35 17 (59) 11 (41) 28

435 5 (71) 2 (29) 7

Exposed to passive smoking 18 (67) 9 (33) 27

Fathers 32 (63) 19 (37) 51

Smoker 25 (66) 13 (34) 38

Biomarkers of PAHs in human bloodNaufal et al.

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

The exposures in mothers and fathers were further

evaluated. A linear regression was analyzed to investigate

the possible correlation between tPAH levels in blood from

paired fathers and mothers (Figure 2a). A correlation that

was statistically significant was detected (correlation coeffi-

cient (r)¼ 0.408; Po0.05). In addition, a linear regression of

the levels of blood tPAHs in paired non-smoking fathers and

mothers revealed a stronger statistically significant correlation

(r¼ 0.662; Po0.05) (Figure 2b).

The association between having a baby with a NTD and

maternal PAH exposure level was examined. Mothers were

divided into two groups, a high exposure group and a low

exposure group, according to their median blood PAH level

(206 ng/ml). Mothers with high PAH exposures were found

to have a significantly higher risk compared with the lower

exposure group (OR 10.91; 95% CI: 2.61–45.60). The age-

adjusted model did not change the increased risk of NTDs

among mothers (OR 8.70; 95% CI: 2.02–37.04) (Table 3).

Bulky DNA adducts were analyzed by 32P-postlabeling in

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

representing mean DNA adduct levels detected in WBCs of

mothers. Mean level of DNA adducts in case mothers was

12.19 per 109 normal nucleotides compared with 17.13 per

109 nucleotides in controls. Similarly, mean level of DNA

adducts in case fathers was 10.97 per 109 nucleotides, which

was significantly lower than 20.64 per 109 nucleotides, the

mean level of bulky DNA adducts in control fathers.

Smoking status did not seem to influence the levels of

DNA adducts in the study participants.

The variable that seemed to have the greatest impact on

DNA adduct levels in mothers was the type of fuel used for

cooking. Mothers reported using types of hard coal, black

coal, firewood, and natural gas as cooking fuels. Hard coal

seems to be the most abundantly used cooking fuel. When

DNA adduct levels in WBCs of mothers who used hard coal

as a cooking fuel were compared with those who use another

type of cooking fuel, they seemed significantly elevated. In

fact, mean levels of DNA adducts in WBCs of mothers using

hard coal were 15 per 109 nucleotides which was significantly

elevated compared with 11 adducts per 109 nucleotides, the

mean level found in WBCs of mothers using other types of

8006004002000

600

500

400

300

200

100

0300250200150100500

500

400

300

200

100

0

N=37r=0.408P<0.05

N=12r=0.662P<0.05

Total PAHs in Venous Blood ofMothers with Non-Smoking Husbands

Total PAHs in Venous Blood of Mothers

To

tal P

AH

s in

Ven

ou

s B

loo

d o

fN

on

-Sm

oki

ng

Fat

her

s

To

tal P

AH

s in

Ven

ou

sB

loo

d o

f F

ath

ers

Figure 2. Linear regression showing the correlation between total PAHs in venous blood from fathers and total PAHs in venous blood frommothers(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.

Table 3. Estimated risk of NTDs based on concentration of PAHsmeasured in whole blood extracts from mothers.

Exposure

levelaCases Controls Crude OR

(95% CI)

Age-adjusted

OR (95% CI)

High 24 3 10.91 (2.61–45.60) 8.70 (2.02–37.04)

Low 11 15

Total 35 18

Abbreviations: CI, confidence interval; OR, odds ratio.aHigh exposure was defined as a blood total PAH level greater than the

median (206 ng/ml); low was defined as a blood PAH level lower than the

median.

0

2

4

6

8

10

12

14

16

18

20

Mothers

*

*P<0.05

CaseN=35

ControlN=18

Mea

n R

AL

per

109

nu

cleo

tid

es

Figure 3. Frequency of bulky DNA adducts (per 109 nucleotides)detected in venous blood from mothers of children born with neuraltube defects (case) and mothers of control children.

Biomarkers of PAHs in human blood Naufal et al.

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

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

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

0

2

4

6

8

10

12

14

16

18

Hard CoalN=34

OtherN=14

Mea

n R

AL

per

109

nu

cleo

tid

es *

*P < 0.05Cooking Fuel

Figure 4. Frequency of bulky DNA adducts in venous blood collectedfrom mothers by the type of fuel used for cooking.

Table 4. Ratio of bulky DNA adducts to carcinogenic PAHs invenous blood based on GSTM1 and GSTT1 polymorphisms

Exposure levela GSTT1 GSTM1

W/t Null W/t Null

High

Mean 0.70 0.63 0.62 0.70

SEM 0.09 0.14 0.11 0.12

N 25 15 23 18

P-value 0.663 0.631

Low

Mean 5.20 4.61 4.02 6.07

SEM 0.76 1.04 0.61 1.10

N 26 12 22 17

P-value 0.656 0.094

aHigh 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.

Biomarkers of PAHs in human bloodNaufal et al.

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

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

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

Biomarkers of PAHs in human blood Naufal et al.

Journal of Exposure Science and Environmental Epidemiology (2010) 20(4) 317

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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