Elevated Levels of PDGF Receptor and MDM2 as Potential Biomarkers for Formaldehyde Intoxication

Formaldehyde has been identified as the most prevalent cause of sick building syndrome (SBS), which has become a major social problem, especially in developing urban areas. However, studies on the molecular mechanisms associated with formaldehyde toxicity have been limited, probably because it is difficult to relate the experimental results obtained from in vitro studies to human exposure in vivo. Using polymerase chain reaction-based suppression subtractive hybridization, we recently identified 27 different formaldehyde-inducible genes including platelet-derived growth factor receptor alpha gene (PDGFRA) and mouse double minute 2 (MDM2) gene which were increased significantly in both formaldehyde-exposed human trachea cells, 680. Tr, and rat tracheas. To establish a possible relationship between induction of these formaldehyde-inducible genes and symptoms of SBS, we examined expression levels of these genes in peripheral lymphocytes of residents of new apartments. Here, we report that the expression of PDGFRA and MDM2 transcripts was significantly higher in peripheral blood lymphocytes obtained from 15 residents in new buildings than in seven control individuals. Our results suggest that the elevated levels of PDGFRA and MDM2 may be associated with the formaldehyde-induced pathophysiology that is closely related with SBS, and that they deserve evaluation as potential biomarkers for formaldehyde intoxication.


INTRODUCTION
Indoor exposure to low levels of noxious chemicals is associated with adverse health effects because people spend a lot of their time indoors (Moschandreas, 1981;Samet et al., 1988). Recently, siek building syndrome (SBS), which is characterized by various symptoms such as respiratory mucosal irritation, skin reactions, unspecific hypersensitivity, mental fatigue, and headache, has become a major social problem. This is especially so in developing urban areas with many newly built apartments (Fernandez-Caldas et al., 1994;Norback et al., 1990). The main causes of SBS are poor indoor air quality caused by insufficient ventilation and the accumulation of noxious compounds such as formaldehyde and volatile organic compounds (Kim et al., 2002;Kilburn, 2000). Of these indoor air chemical pollutants, formaldehyde has been identified as the most prevalent, probably because it is very often used for glued wood products in residential environments, such as particleboard, floor coverings, and furniture (Kilburn, 2000;Main and Hogan, 1983). Indeed, indoor formaldehyde concentrations in 46.7% of new apartments in Korea were higher than the 100 flg/m3 that is the Japanese safety standard (NIER, 2004).
Formaldehyde has been considered as a potent respiratory and conjunctival irritant (Godish, 1990;Hendrick et al., 1982). Although indoor formaldehyde exposure is hazardous to human health, studies on the molecular mechanisms associated with formaldehyde toxicity have been limited, probably because it is difficult to relate the experimental results obtained from in vitra studies to human exposure in vivo. Recently, we identified 27 different formaldehyde-inducible genes that could be associated with the biological effects of formaldehyde (Lee et al., 2008). To establish a possible relationship between formaldehyde-induced gene expression and symptoms of SBS, we examined the expression levels of these genes in peripheral Iymphocytes of residents of new apartments in comparison with results from control resi-dents of old apartments. Among these genes, expression levels of those for PDGFRA and MDM2 were increased significantly in the peripheral Iymphocytes of new building residents. Thus, elevated expression levels of these genes could be candidate bio markers for formaldehyde toxicity.

MATERIALS AND METHODS
Blood collection from human subjects. Twentytwo volunteer subjects, 15 residents of new apartments and seven control residents of old apartments, were recruited from Kongju, located in Chungnam province in Korea for this study. Approval for the study was obtained from the Standing Committee for Ethics in Research on Humans at Seoul National University. Brief information sheets were distributed among the residents of newly constructed apartment to seek volunteers for this study. For measurements of indoor formaldehyde concentration, air sampies were collected after an unannounced visit in May 2006. The concentration of formaldehyde in the indoor air was monitored by formaldehyde meter (W/Pump Model Z-300XP). Whole blood sam pies were collected from the experimental subjects into heparin-coated tubes, mixed with RNAlate~ (Ambion Inc., Ausrin, TX) and stored at -20°C until use.
Semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Total RNA was isolated using the RiboPure-Blood® Kit (Ambion Inc.) according to the manufacturer's protocols. Isolated sam pies were treated with DNase land single-stranded cDNA was synthesized from the RNA in areaction mixture containing random hexamers and 200 U of murine Moloney leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) (Shin et al., 2003). The PCR reaction was performed with specific primers, as previously described (Lee et al., 2008). The genes were analyzed under conditions in which their PCR products were amplified exponentially.
Statistical analysis. Experimental values are expressed as the mean ± S.D. The significance of any difference was determined by Student's t tests and expressed as a probability value. Mean differences were considered significant at P< 0.05.

RESULTS
Formaldehyde is a ubiquitous chemical toxicant that probably causes SBS: adverse health effects caused by indoor air pollution in residents of newly constructed buildings. Recently, we identified 27 different formaldehyde-inducible genes that could be associated with the biological effects of formaldehyde (Lee et al., 2008). To establish a possible relationship between formaldehydeinduced gene expression and symptoms of SBS, we examined the expression levels of genes for calcyclin, glutathione S-transferase pi, PDGFRA, MDM2 and HLA-A in peripheral Iymphocytes of 15 residents of new apartments, and these were compared with results from seven control residents of old apartments. Details regarding the experimental subjects are described in Table 1. The mean indoor formaldehyde levels in the bedroom of old and new apartments were 0.027 ± 0.016 ppm and 0.311 ± 0.33 ppm, respectively; thus they were significantly higher in the new apartments (Fig. 1). The formaldehyde concentrations in new apartments ranged from 0.01 to 1.07 ppm, and nine sampies (60%) exceeded 0.08 ppm, the current Japanese safety standard. To examine expression levels of formaldehydeinducible genes, semiquantitative RT-PCR analysis was performed using mRNA obtained from whole blood sampies. Intensity of the PCR bands was largely var--E c.. c.. ied for PDGFRA and MDM2 in experimental individuals (Fig. 2). Quantifieation of the PCR bands showed that the expression levels of PDGFRA and MDM2 were inereased approximately 1.9-fold and 2.3-fold, respeetively, in the residents of new apartments to eompare with the residents of old apartments (Fig. 3). However, the expression of other genes, such as those for caleyclin, GSTP1 and HLA-A, were not mueh altered.

DISCUSSION
Formaldehyde is a ubiquitous ehemical toxicant that probably eauses SBS: adverse health effects caused by indoor air pollution in residents of newly construeted buildings. Here, we report that the expression of PDG-FRA and MDM2 transeripts was significantly higher in peripheral blood Iymphoeytes obtained from 15 residents in new buildings than in seven eontrol individuals. Our results suggest that the elevated levels of PDGFRA and MDM2 may be associated with the formaldehyde-induced pathophysiology that is closely related with SBS, and that they deserve evaluation as potential biomarkers for formaldehyde intoxication.
PDGFRA and PDGFRB are expressed in eosinophils and their signaling is involved in eosinophil funetion (Adaehi et al., 2006). Inereased indoor eoneentrations of formaldehyde are associated with asthma, one of the most eommon respiratory diseases. These allergie symptoms indueed by formaldehyde are eharaeterized by inflammatory cell infiltration, mostly eosinophils, whieh are the major inflammatory eells of allergie reaetions aetivated by respiratory mueosal damage (Casset et al., 2006;Ohtsuka et al., 2003). Therefore, the inerease in PDGFRA level may eontribute to the allergie symptoms caused by low-Ievel exposure of air-born formaldehyde in the new apartments. The MDM2 oneoprotein is a primary regulator of the p53 tumor suppressor gene, because it eontrols protein level of p53 via ubiquitin-dependent proteasomal degradation. Exposure to formaldehyde increases the level of DNA damage characterized by DNA-protein crosslinks (DPC) (Heck et al., 1990;Heck and Casanova, 1999;Shaham et al., 2003).
DPC increased mutations in p53 in peripheral blood Iymphocytes of human after formaldehyde exposure, suggesting that DPC and mutation in p53 may represent steps in formaldehyde-induced carcinogenesis (Shaham et al., 2003). Our results may suggest that the induction of MDM2 expression may indicate a possibility of induction of p53 expression and DNA damage after formaldehyde exposure in SBS.
In the USA, the mean concentration of indoor formaldehyde has been reported as 38 flg/m3 (30 ppb), with a typical range of 13-350 flg/m3 (Stenton and Hendrick, 1994). In Australia, an indoor guideline of 125 flg/m3 (100 ppb) has been set (McPhail, 1991). Irritant effects of the upper respiratory tract are a well-established consequence of formaldehyde exposure above 150 flg/m3 (Stenton and Hendrick, 1994). Formaldehyde exposure at levels weil below 100 ppb may lead to an increased risk of allergie sensitization to common aeroallergens in children (Garrett, 1999). We found that indoor levels of formaldehyde in newly built apartments were substantially higher (310 ppb) than the present guidelines of 100 ppb in Australia and 80 ppb in Japan (McPhail, 1991). Importantly, these changes in PDGFRA and MDM2 expression patterns in residents of new buildings are consistent with our observations in this animal model in which relatively higher levels of formaldehyde were applied (3 ppm and 38 ppm) (Lee et al., 2008).
These results suggest that the low-Ievel exposure to formaldehyde of new building residents may have a potential to cause the pathological symptoms of epithelial damage and allergie inflammation that was seen in following the high level exposure in rats. Taken together, indoor guidelines for formaldehyde exposure may need to be reviewed in light of these findings.
In summary, we observed that the expression levels of PDGFRA and MDM2 were increased significantly in the peripheral blood Iymphocytes of the residents of newly built apartments, suggesting that these genes may be associated with the pathologies induced by formaldehyde exposure during SBS. Our findings on gene expression can be applied in follow-up programs of residents of new buildings exposed to formaldehyde to identify high risk populations and to help preventing SBS.