Chinese-German Journal of Clinical Oncology March 2012, Vol. 11, No. 3, P146–P148DOI 10.1007/s10330-011-0931-3

Polycyclic aromatic hydrocarbons (PAHs) is defined as a compound with two or more benzene rings arranging in the way of linear, angular or cluster-like. Carcinogenic, teratogenic and mutagenic potential are the prominent features of the PAHs. The pollution problems and pre-ventive measure about PAHs has became one of the is-sues urgently to be solved these years [1, 2]. Previous study found that long-term exposure to PAHs is closely related with the occurrence of pancreatic cancer [3]. So we sup-pose the PAHs content in human pancreatic cancer tis-sues is observable and it’s one of the pathogenic factors in pancreatic tumorigenesis.

This study was designed to detect the content of the PAHs in human pancreatic tissues (cancer tissue and non-cancerous tissue) with the method of ultrasonic ex-traction (UE), solid phase extraction and clean up-high performance liquid chromatography with fluorescence spectroscopy. Then we can confirm whether the PAHs exist in human pancreatic cancer tissues whether it’s re-sponsible with pancreatic tumorigenesis.

Materials and methods

The main reagentsBenzo (a) pyrene, pyrene, phenanthrene, 2–methyl

anthracene, fluoranthene, chrysene, 2-methylnaphtha-lene were spectroscopically pure reagent (purchased from Fluka Company, USA). Methanol, hexane, dichlorometh-ane, acetonitrile, anhydrous sodium sulfate were domes-tic analytical reagent.

InstrumentsLC-240 high performance liquid chromatography

(HPLC; Perkin–Elmer Company, USA).Solid-phase extraction (SPE) device (Dalian Institute

of Chemical Physics, Chinese Academy of Sciences).H66025 ultrasonic bath (Wuxi Ultrasonic Electronic

Equipment Factory, China).ISOLUTE SPE-C18 Columns (Fuji Technology Co.,

Ltd., China).

The preparation of the eight kinds of PAHs’ standard solution

Eight kinds of PAHs prepared with methanol as the solvent were used for the study: naphthalene: 50 μg/mL; 2-methylnaphthalene: 50 μg/mL; phenanthrene: 10 μg/

The detection and significance of polycyclic aromatic hydrocarbons in human pancreatic cancer*Shuntao Liu, Limin Lun

Clinical Laboratory, The Affiliated Hospital of Medical College Qingdao University, Qingdao 266061, China

Received: 18 November 2011 / Revised: 5 January 2012 / Accepted: 15 January 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The aim of this study was to investigate the effect of polycyclic aromatic hydrocarbons (PAHs) on the occurrence of human pancreatic cancer. Methods: PAHs in human pancreatic cancer, adjacent pancreatic cancer tissues and tissues without pancreatic cancer were extracted by ultrasonic extraction (UE). And then the extracts were cleaned up by solid phase extraction and analyzed by high performance liquid chromatography (HPLC) with fluorescence spectroscopy. Results: Four kinds of PAHs were detected, which were chrysene, 2-methylanthracene, pyrene and benzo (a) pyrene. The contents of the four PAHs were not statistically significant between pancreatic cancer and adjacent tissues (P > 0.05). The contents of 2-methylanthracene, pyrene and benzo (a) pyrene in pancreatic cancer and adjacent tissues were higher than tissues without pancreatic cancer, the differences were statistically significant (P < 0.05). The contents of chrysene in the three kinds of pancreatic tissues were not statistically significant (P > 0.05). Conclusion: PAHs were found in human pancreatic tissues. Human pancreatic tissues have extremely strong ability of bio-concentrating PAHs. PAHs might play an important role in the occurrence of human pancreatic cancer.

Key words pancreatic neoplasms; polycyclic aromatic hydrocarbons (PAHS); high performance liquid chromatography (HPLC)

Correspondence to: Limin lun. Email: lunlm@yahoo.com.cn* Supported by a grant from Key Laboratory of Marine Spill Oil Identifica-tion and Damage Assessment Technology (No. 200902).

147Chinese-German J Clin Oncol, March 2012, Vol. 11, No. 3

mL; fluoranthene: 5.0 μg/mL; pyrene: 5.0 μg/mL; 2-meth-ylanthracene: 5.0 μg/mL; Chrysene: 5.0 μg/mL; Benzo (a) pyrene: 1.0 μg/mL. The standard solutions were frozen at -20 ℃ for later use. They can be diluted to the appropri-ate concentration when used.

Specimen source and groupsSpecimens were collected from Department of Pathol-

ogy, the Affiliated Hospital of Medical College Qingdao University, China, and confirmed by pathology. The ex-periment was divided into three groups: the human pan-creatic cancer tissues [18 cases, 11 males and 7 females, aged (60.2 ± 10.1) years], the adjacent tissues [18 cases, 11 males and 7 females, aged (60.2 ± 10.1) years], and pan-creatic tissues without pancreatic cancer [6 cases, 4 males and 2 females, aged (40.1 ± 15.8) years]. There was no statistical significance in age, sex ratio among the three groups.

Extraction and purification of PAHs in pancreatic tissues

The specimens were mashed into a paste. Assay sample (1g) was inserted in a glass tube with 30 mL hexane–di-chloromethane (1:1) and placed in an ultrasonic bath for 10 min. The solution was centrifuged for 5 min at 2500 rpm and the last two steps were repeated twice. The sonicated extracts were evaporated in a rotary vacuum evaporator to almost dryness (approximately to 0.5 mL) for further clean up. The extracts were purified follow-ing a clean up procedure using solid-phase extraction cartridges of neutral alumina of 5 g ISOLUTE. The alu-mina was solvated and conditioned prior to sample load-ing with 20 mL hexane-dichloromethane (2:1) and 20 mL hexane-dichloromethane (10:1). The extract was added to the top of the column and analyte elution was per-formed with 100 mL hexane-dichloromethane (10:1) and afterwards with 100 mL hexane-dichloromethane (2:1). The fractions were collected, pre-concentrated in a ro-

tary vacuum evaporator to 0.5 mL and transferred into vials. Extracts were evaporated at room temperature with a gente stream of nitrogen and reconstituted in 250 μL of acetonitrile.

Chemical analysisTwenty microliters of the final samples were inserted

three times into a high-performance liquid chromatog-raphy (HPLC) system. A PAH column, coupled to pho-to diode array (PDA) and fluorescence detectors (FLD) and acetonitrile-water isocratic (87:13) mobile phase at 25℃ were used. Conditions: mobile phase at 1 mL/min, degassing by helium 50 mL/min. To increase sensitivity, fluorescence wave length programming was used during chromatographic separation. Individual PAHs were iden-tified on the basis of retention time by using a process-ing method and library, while their concentrations were determined with the use of calibration curves and peak area ratios of the analyte peak to the area of the internal standard peak.

Statistical analysisAll values were expressed as mean ± standard error.

PPMS 1.5 statistics software application data processing, variance analysis, significant level α = 0.05.

Results

Chromatogram of eight kind of PAHs standard solu-tion was presented in Fig. 1. Detection rate of PAHs in the three pancreatic tissues were reported in Table 1. PAH contents determined in three pancreatic tissues were reported in Table 2. Four kinds of PAHs were de-tected in human pancreatic tissues which were chrysene, 2-methylanthracene, pyrene and benzo (a) pyrene. The contents of the four PAHs were not statistically signifi-cant between pancreatic cancer and adjacent tissues (P > 0.05). The contents of 2-methylanthracene, pyrene and

Table 1 Detection rate of PAHs in the three pancreatic tissues (n, %)

Groups n Chrysene Pyrene 2-methylanthracene Benzo (a) pyrenen % n % n % n %

Pancreatic cancer tissues 18 16 89 18 100 16 89 18 100Adjacent pancreatic cancer tissues 18 18 100 18 100 15 78 18 100Tissues without pancreatic cancer 6 4 67 6 100 3 50 6 100

Table 2 Concentrations of PAHs in the three pancreatic tissues (ng/g; χ ± s)Groups Chrysene Pyrene 2-methylanthracene Benzo (a) pyrenePancreatic cancer tissues 326.2 ± 201.8 559.6 ± 241.5 379.7 ± 185.7 629.3 ± 333.9Adjacent pancreatic cancer tissues 398.7 ± 183.5 542.3 ± 225.6 320.2 ± 137.8 598.3 ± 245.5Tissues without pancreatic cancer 309.4 ± 153.5** 239.4 ± 200.1* 113.3 ± 98.6* 255.2 ± 105.4*** F = 0.87, q (0.267 7–1.633 7), P > 0.05; * F (4.39–6.51), pancreatic cancer tissues and adjacent pancreatic tissues were compared with tissues with-out pancreatic cancer q (3.726 5–5.100 1), P < 0.05, pancreatic cancer tissues compared with the adjacent tissues q (0.319 6–1.610 9), P > 0.05

148 www.springerlink.com/content/1613-9089

benzo (a) pyrene in pancreatic cancer and adjacent tissues were higher than tissues without pancreatic cancer, the differences were statistically significant (P < 0.05). The contents of chrysene in the three kinds of pancreatic tis-sues were not statistically significant (P > 0.05).

Discussion

PAHs has a trace content in the environment, but epi-demiological studies [4, 5] show that PAHs is endued with a magnified biological function and toxic effect as a re-sult of biologically cumulative effect in food chain. In our previous studies [6, 7], we detected PAHs in human gastric cancer and lung cancer, and all the cancer tissues had a significantly higher content than the non-cancer ones. Yang et al [8] found that the amount of PAHs produced by indoor coal-fired pollution may be the main risk factor of Xuanwei female lung cancers. In this study, we proved that PAHs can be detected in human pancreatic cancer tissues and it might play an important role in the develop-ment and progression of human pancreatic cancer.

PAHs is thought to be activated by CYP4501A1, a member of cytochrome P450 enzyme system (CYP450s), and then evolved into carcinogenic electrophilic epoxides after enter the body. So the activity of CYP4501A1 isoen-zyme becomes the key to decide the carcinogenicity of PAHs [9]. The activity of CYP4501A1 isoenzyme can be induced by PAHs, and the activity increases significantly in peoples with long-term exposure to PAHs [10]. Previous study indicated that people with high PAHs inductivity genotype had a higher risk of suffering from colorectal cancer and lung cancer than non-high-inductivity geno-type [11]. But no definite mechanisms of PAHs’ carcinoge-nicity were founded in our study and farther experiments about this are needed.

By present, all the detections about PAHs were lim-ited in environment, food, animals and plants, and few researches about the human body tissues can be founded. In this study, we proved that PAHs existed in human pan-creatic tissues with the methods of UE, SPE and HPLC. Further analysis showed that PAHs content were higher in pancreatic cancer tissues than non-cancer ones, and there was a significant difference between each other (P < 0.05). This indicated that PAHs may be associated with the occurrence of pancreatic cancer and it may be one of the most important reasons to generate human pancreatic cancer. Therefore, controlling PAHs contamination and reducing PAHs exposure have an important meaning for pancreatic cancer prevention.

References

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Fig. 1 Chromatogram of eight kind of PAHs standard solution. 1: naph-thalene; 2: 2-methylnaphthalene; 3: Philippines; 4: pyrene; 5: 2-methylan-thracene; 6: fluoranthene; 7: chrysene; 8: benzo (a) pyrene