BLOOD AHR ACTIVATOR LEVELS AND AHR MESSENGER RNA EXPRESSION IN
POPULATIONS WITH HIGH AND BACKGROUND EXPOSURE TO PCP WASTE
Lin S1, Tsai S-F1, Wang S-L1, Nakamura M2, Handa H2, Sundaram P2, Liou S-H1
1

National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County, Taiwan; 2Hiyoshi
Corporation, 908 Kitanoshocho, Omihachiman, Shiga 523-8555, Japan.

Introduction
The toxic and biological effects of TCDD and dioxin-like compounds are mediated by aryl hydrocabon receptor(AhR)1. Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs),polyaromatic hydrocarbons (PAHs), polychlorinated naphthalenes (PCNs), hexachlorobenzene, and derivativesof these compounds can also activate AhR2. AhR-dependent toxicity appears to be primarily driven by abnormal
and persistent activation of AhR-dependent gene expression in target cells3. Natural and endogenous ligands ofAhR -including flavonoids, carotenoids, tryptophan, and arachidonic acid metabolites- have relatively weakaffinity compared to TCDD and are rapidly degraded 4. Human exposure to PCDD/Fs and PCBs is associatedwith serious consequences to health including developmental5,6, neurological7, immunological8, reproductive9-12,
and carcinogenic effects13,14.
Pentachlorophenol (PCP) and chemical waste produced by a factory in southwestern Taiwan continued tocontaminate the surrounding area even after the plant closed. Sera PCDD/PCDF levels of residents in thecontaminated area have been found to be higher than those of non-contaminated area. These residents may havehigher AhR ligand levels in their blood, which would probably raise the AhR activity abnormally and might leadto related health effects.
In this work we measured AhR activators with an alternative clean-up method conserving PAH, in order tomaximize the estimation of the biological dose of AhR activators in blood samples of residents from the above
mentioned contaminated area and other non-contaminated areas. We also correlated the Calux results with theAhR mRNA expression in the blood samples of these residents.

Materials and methods
A total of 504 subjects, 271 residents of the high exposure area (Annan District, Tainan) and 210 from a nonexposure area serving as reference group were recruited for the study.
Clean-up and CALUX were performed at Hiyoshi Corporation, Japan. Lipid fraction of 10 mL of whole blood
with heparin was extracted with acetone and n-hexane, cleaned up using a celite-sodium sulphate column, dried
with evaporator and transferred to silica-gel columns in hexane, without sulphuric acid silica gel layers, to retain
PAH.
Thereafter H1L6.1 cell line, a mouse hepatoma (Hepa1c1c7)-derived cell line which has been stably transfectedwith the DRE-driven firefly luciferase reporter plasmid (pGudLuc6.1), was used for the CALUX system with 20hours of incubation. CALUX results were expressed as relative luciferase activity (RLA), RLA per gram fat(RLA/g fat), and RLA per gram blood (RLA/g blood). Luciferase activity of 1 nM TCDD has RLA=1.
Buffy coat was obtained from blood anticoagulated with EDTA for AhR mRNA determination. After washingwith red blood cells lysis buffer, white blood cells were kept in Trizol. Total RNA was isolated from white bloodcells using with Acid Phenol-Guanidium thiocyanate- chloroform extraction method. Convertion to cDNA wasperformed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). AhR and GAPDH

Organohalogen Compounds

Vol. 73, 1594-1595 (2011)1594

messenger RNA were measured using the ABI PRISM 7900HT Sequence Detection System with Taqman
Hs00169233_m1 and Hs99999905_m1.
Statistical analyses were performed with SPSS 17.0.

Results(1) Exposure area residents had relatively higher AhR activator levels than control area residents (average =144.47 vs. 72.43 pg TEQ/g fat). After controlling for age, BMI, and smoking status, the differences werestill statistically significant, either considering both genders together or separately.
(2) AhR expression (normalized with GAPDH) was also higher in the exposure area (1.03±0.05) compared tothe control areas (0.83±0.03) (t=3.293, p<0.001).
(3) Significant correlation (Spearman’s rho 0.207, p<0.02) between AhR ligand levels as determined withCALUX and AhR expression in terms of mRNA was found.

Conclusions
We have found higher levels of total AhR ligands and AhR mRNA expression in bloods of residents from a PCP
contaminated area compared to other non-contaminated areas. Blood AhR ligand levels and AhR mRNA
expression was associated, suggesting that the AhR ligands may be inducing AhR expression. Whether abnormal
AhR expression would to AhR related health consequences would be our next subject of study.

Acknowledgements
We are thankful to the National Science Council (NSC 97-2314-B-400-007-MY3), Taiwan and Hiyoshi
Corporation for supporting this research.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.

Safe S.Toxicology Letters 2001; 120(1-3): 1
Janosek J., Hilscherova K., Blaha L., Holoubek I. Toxicol In Vitro 2006; 20(1): 18
Zhao B., Bohonowych J.E., Denison M.S. Organohalogen Compounds 2006; 68:1745
Denison M.S., Pandini A., Nagy S.R. Baldwin E.P., Bonati L. Chemico-biological Interactions 2002;
141(1-2): 3Ulbrich B., Stahlmann S. Arch Toxicol 2004; 78(5): 252
Walkowich J., Wiener J.A., Fastabend A., Heinzow B., Kramer U., Schmidt E., Steingruber H. J.,
Wundram S., Winneke G. Lancet 2001; 358 (9293):1602
Jacobson J.L., Jacobson S.W. Neurotoxicology and Teratology 2002; 24(3):349
Lindstrom G., Hooper K., Petreas M. Stephens R., Gilman, A. Environmental Health Perspectives 1995;
103 Supppl 2: 135
Gray L..E Jr and Ostby J.S. Toxicology and aplied pharmacology 1995; 133(2):285
Guo Y.L., Hsu P.C., Hsu C.C., Lambert G. H. Lancet 2000; 356 (9237):1240
Mably T.A., Moore R.W., Peterson R.E. Toxicology and applied pharmacology 1992;114(1):97
Mably T.A., Bjerke D.L.,Moore R.W. Gendron-Fitzpatrick A., Peterson R.E. Toxicology and applied
pharmacology 1992; 114(1): 118
Schecter A., Olson J.R., Chemosphere 1997; 34(5-7):1569
Walker N.J.,Crockett P.W., Nyska A., Brix A.E., Jokinen M.P., Sells D.M., Hailey J.R., Easterling M.,
Haseman J.K., Yin M., Wyde M.E., Bucher, J.R., Portier C.J. Environmental Health Perspectives 2005;
113(1): 43

Organohalogen Compounds

Vol. 73, 1594-1595 (2011)

1595