Abstract
Polycyclic Aromatic Hydrocarbons (PAHs) are the large group of organic compounds with benzene rings that are mainly emitted during incomplete combustion of organic materials such as fossil fuels. Many PAHs are linked to carcinogenicity and mutagenicity and their presence in ambient air is an increasing day by day particularly in developing countries. In urban regions, more number of people are exposed to the PAHs since the emission sources of PAHs are many including vehicular emissions. Understanding the health impacts of PAHs in human beings is one of the challenging tasks which include laborious experiments. As an easy approach, biomarkers of PAH exposure offers an immediate insight into exposed population by simple methods. In the present study, we analyzed urine samples of 30 volunteers in three different cities of Southern India for PAH metabolites that are considered as potential biomarkers for PAH exposure. The presence of 2-hydroxynaphthalene (2-NAP) and 1-hydroxypyrene (1-OHP) in majority of samples indicated that a substantial population might be exposed to PAHs.
Keywords: PAH; Biomarkers; Urinary Metabolites
Introduction
Biomarkers have been increasingly recognized worldwide for exposure assessment of hazardous chemicals. Particularly in exposure assessment the exogenous substance or first metabolite or the product of an interaction between a xenobiotic agent and some target molecule or cell that is measured as an indicator of an inherent or acquired ability of an organism to respond to the challenge of exposure to a specific xenobiotic substance [1]. In many cases, biomarkers can also serve as indicators of preclinical conditions, and demand the need for a detailed assessment. Biomarker assessment is also considered as advantageous and quick technique over the laborious dose-effect studies. Recent attention in epidemiology focuses on understanding the genetic basis for individual susceptibility to the development of chronic disease and interaction with the biomarkers of exposure [2]. Genetic susceptibility markers are very useful for addressing gene– exposure (i.e., gene–environment) interactions.
In evaluating the health effects and risks of PAH, great advances have been achieved in the development of various types of biomarkers, including chemical-specific biomarkers of exposure and characterization of genetic variation in many genes of relevance to carcinogenesis [3]. The most commonly used biomarkers of PAH exposure includes hydroxylated PAH metabolites in urine (OHPAHs, 2-naphthol, 2-hydroxyfluorene, 3-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene and 3-hydroxybenzo[a] pyrene) [4].
The assay for the glucuronide conjugate of 1-OHP (1- OHPG) has been developed and successfully applied to population with various PAH exposures. 1-OHPG is more sensitive than 1-OHP, since 1-OHPG is 3–5 times more fluorescent than 1-OHP. On the other hand, the 1-OHPG assay requires more urine than the 1-OHP assay. Urinary 2-naphthol, a stable PAH metabolite, reflects more specifically ambient PAH exposure, whereas 1-OHP levels can be affected by diet and smoking. Thus, urinary 2-naphthol is suggested as a specific marker for exposure to airborne particulates whereas urinary 1-OHP has been used as a marker for exposure to PAHs by non-specific exposure routes [2].
While evaluating the occupational exposure of PAHs, [5] observed that waste incineration workers showed higher level of 1-OHP and 2-naphthol than the automobile emission inspection workers in Korea. The levels of 1-OHP in automobile emission inspectors, waste incineration workers and control subjects were 0.298 ± 0.212, 0.531 ± 0.427 and 0.061 ± 0.094 μmol/mol creatinine, respectively. The mean values of 2-naphthol were 5.894 ± 4.683, 8.947 ± 5.931 while compared to control 1.924 ± 3.441 mol/mol creatinine, which ascertained that biomarker is a potential tool for exposure studies. In contrary to the belief that inhalation as a major route of exposure to PAHs, [6] in a study at Japan identified dietary exposure was significantly correlated with urinary 1-OHP excretion than inhalation. Biomarker experiments also revealed that the average concentrations of 1-OHP in the urine samples were higher smokers and passive smoker groups than that of the non-smoking group [7].
Zheng et al (2013) suggested hydroxylated PAH metabolites (OH-PAHs) as suitable biomarkers for wood smoke exposure. In an experiment involving nine volunteers, urinary OH-PAHs, except 1-hydroxypyrene (1-PYR), correlated with those of PM 2.5, levoglucosan and PAHs in personal PM 2.5 samples. In the children subjects also urine samples appeared to be the best biomarkers; a study in Ohio at 126 homes and 16 daycares observed associations between selected sociodemographic/lifestyle factors and urinary 1-OHPyr levels [8,9]. The median urinary 1-OHPyr level was 0.33 ng/ mL. Therefore, large number of studies on biomarkers have widely recognized PAH urinary metabolites as potential biomarkers for human exposure studies.
Materials and Methods
Urine samples (early morning) from 30 volunteers (10 each from 3 cities: Chennai, Coimbatore and Tiruchirappalli; Figure 1) were collected and analyzed for urinary PAH metabolites. During the sample collection, volunteers were requested information on health and living habits, such as age, height, weight, disease prevalence and smoking habits. Early morning urine samples were collected in bottles pre-cleaned with deionized water and 0.1 M Hydrochloric acid (HCl). The samples were taken immediately to the laboratory. Volunteer’s age were between 23 to 45 years, of which 80% are males and 20% females. All of them resided in urban environments
Figure 1: Study Area.
Urinary metabolite was analyzed according to the method of [10]. In brief 10 ml of urine was buffered with 20 ml of acetate buffer and the metabolite was deconjugated by 15 μl of β-glucuronidase/arylsulfatase (type H-2, Sigma, USA) at 37°C. The hydrolyzed urine sample was then loaded on to the SEP-Pack C18-E cartridge (Phenomenex-USA) at a flow rate of <1 ml/min. The column was washed with 10 ml water and 10 ml 30% methanol to remove the matrix interferences. The two PAHs were eluted with 4ml of methanol. Elute was concentrated almost to dryness under a gentle stream of nitrogen, and then dissolved in 1 ml of methanol. The solution was filtered through 0.2 μm filter, and then stored at -20deg;C until analysis.
Two PAH metabolites [2-hydroxynaphthalene (2-NAP) and 1-hydroxypyrene (1-OHP)] standards obtained from SUPELCO were used to generate standard chromatogram and the optimum wavelengths were standardized. Analysis was carried out with a HPLC-FLD (Make: Waters) with PAH C18 column (5 μm 4.6 x 250 mm). Elution was performed using a water–methanol gradient: 0-15 min a linear gradient from 50% to 70% methanol and 15-20 min a linear gradient from 70% to 90% methanol. The flow rate was 0.80 ml/ min. The volume of sample injection was 20 μl.
Result and Discussion
Urinary 1-OHP and 2-NAP, the biological markers used for analyzed of PAH exposure analysis is shown in Figure 2.
Figure 2: Chemical structure of PAH metabolites.
Ten volunteers selected from Chennai city were in the age group 20-30. Majority of them were students while three people were employed in different occupation Table 2. Two urinary PAH metabolites viz. 2-NAP and 1-OHP were observed with maximum values of 76.08 ng/l and 22.11 ng/l, respectively. The level of 1-OHP and 2-NAP in general was observed higher in smokers. Although no conclusion about PAH exposure can be arrived with limited number samples, these results hint that a substantial number of population might be exposed to higher levels of PAH.
Time (min)
Excitation wavelength (nm)
Emission wavelength (nm)
PAH metabolites
0-14
227
355
2-hydroxynaphthalene (2-NAP)
17-22
242
388
1-hydroxypyrene (1-OHP)
Table 1: The optimal excitation and emission wavelengths for two PAH metabolites.
Height (cm)
Weight (kg)
Occupation
mode of travel
Smoking habits
2- Naphthol ng/L levels
1-OHP ng/L levels
168
54
Student
walk
No
16.69
2.976
172
70
Student
walk
No
BDL
2.878
165
64
Student
walk
No
26.41
11.74
195
99
Student
Two Wheeler
Yes
34.85
22.11
160
55
Student
Train
No
BDL
BDL
180
78
Foundry
Train
No
76.08
2.288
155
60
Student
walk
Yes
15.82
BDL
156
55
IT Professional
Train
Yes
24.05
1.13
164
60
Press
Train
Yes
28.06
0.756
174
71
Student
walk
Yes
BDL
BDL
BDL: Below detectable levels
Table 2: Urinary PAH metabolite levels of 10 individuals in age group 30-40 and their background in Chennai.
In Coimbatore city, 10 respondents in the age group 26-52 were examined. Urinary metabolites such as 2-NAP and 1-OHP concentration were observed up to the level of 93.07 and 10.45 ng/l respectively Table 3. Sample of police man recorded highest concentration of 2-NAP (93.07 ng/l) indicating clearly is his occupational exposure to traffic emissions. In general, level of urinary metabolites was high in male, particularly smokers. Variation in biomarker levels among the respondents may be due to extent of exposure and other influencing factors such as smoking, body mass index and diet.
Height (cm)
Weight (kg)
Occupation
mode of travel
Smoking habits
2- NAP ng/L
1-OHP ng/L
154
51
Homemaker (Female)
Bus
No
BDL
BDL
172
75
Clerk (Male)
Two wheeler
Yes
12.55
0.71
172
75
Police (Male)
Four wheeler
Yes
93.07
1.08
174
62
Farmer (Male)
Bus
Yes
30.03
BDL
171
75
Engineer(Male)
Two wheeler
No
9.47
BDL
160
45
Merchant (Male)
Bus
Yes
BDL
10.45
158
51
Clerk (Male)
Two wheeler
No
15.3
BDL
150
48
Homemaker (Female)
Bus
No
BDL
1.88
155
70
Homemaker (Female)
Bus
No
41.84
BDL
162
51
Student (Male)
Bus
Yes
BDL
8.92
Table 3: Urinary PAH metabolite levels of 10 individuals in Coimbatore of age group 26-52 and their background.
In Tiruchirappalli, among the 10 Urine samples analyzed for 1-OHP and 2-Naphthol, Higher levels of 2-NAP concentrations ranging between 36-96 ng/l was recorded in half the number of samples. 1-OHP levels recorded up to 6.76 ng/l in smokers Table 4.
Height (cm)
Weight (kg)
Occupation
Mode of travel
Smoking habits
2- Naphthol (ng/L)
1-OHP (ng/L)
174
64
Business
Two Wheeler
No
67.17
1.98
162
55
Student
Two Wheeler
Yes
BDL
3.46
160
72
Student
walk
Yes
96.93
0.42
175
78
Student
Two Wheeler
No
BDL
0.42
160
58
Student
Bus
Yes
55.89
6.76
180
78
Sales man
Two Wheeler
Yes
36.8
1.67
158
65
Student
Walk
No
BDL
BDL
164
54
Sales man
walk
Yes
BDL
0.3
165
78
Business
Two Wheeler
Yes
BDL
2.65
170
71
Student
walk
No
12.34
2.16
Table 4: Urinary PAH metabolite levels of 10 individuals in Coimbatore of age group 26-52 and their background.
Conclusion
The excess levels of two urinary PAH metabolites (2-NAP and 1-OHP) observed in majority of respondent hints that possibility of higher population being exposed to PAHs. With the current trend in urbanization and vehicular growth more population will be at risk of PAH exposure. Future urban growth should be inclusive facilities for environmentally benign garbage disposal, traffic decongestion plans and eco-friendly fuel driven public transport systems. Rewarding experiences from western countries such as introduction congestion tax, technology retrofit, and stringent emission norms can also be explored in Indian cities.
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