Faculty Bibliography

The number of perfluorochemicals (PFCs) that have been found in biological and environmental matrices is increasing as analytical standards and methods evolve. Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) constitute only a fraction of the total suite of PFCs found in environmental and biological matrices. A robust method and approach is needed to evaluate the mass of fluorinated compounds in biological matrices. In this study, we developed a method to measure total fluorine (TF) and organic fluorine (TOF) in human blood matrices using combustion ion chromatography (CIC). Blood matrices (whole blood, serum, and plasma) were analyzed in bulk to determine TF. An aliquot of the blood was also extracted with organic solvents such as methyl-tert-butyl ether (MTBE) and hexane, and organic and aqueous extracts were separated, to fractionate organofluorines from inorganic fluorine. The organic layer was analyzed for TF by CIC, and for known PFCs by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). PFCs measured by HPLC-MS/MS accounted for >80% of the TF in the organic fraction. The aqueous fraction contained inorganic fluorine and other non-extractable organofluorines. However, in the bulk sample, fluoride and non-extractable organofluorines accounted for >70% of the TF in blood samples from the general population. In occupationally exposed individuals, known organofluorines accounted for a major proportion of the TF. These results suggest the existence of yet uncharacterized fluorine fraction in human blood. Further studies are needed to characterize the aqueous fraction that contains inorganic fluorine and non-extractable forms of fluorine.

Synthetic musk compounds are used as additives in many consumer products, including perfumes, deodorants, and detergents. Earlier studies have reported the occurrence of synthetic musks in environmental and wildlife samples collected in the United States. In this study, human breast milk samples collected from Massachusetts, were analyzed for the determination of concentrations of synthetic musks such as musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene), musk ketone (4-tert-butyl-2,6-dimethyl-3,5-dinitroacetophenone), HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta[gamma]-2-benzopyran), AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene), and HHCB-lactone, the oxidation product of HHCB. In addition, we estimated the daily intake of synthetic musks by infants based on the ingestion rate of breast milk. Synthetic musks were found in most of the samples analyzed, and the concentrations ranged from < 2 to 150 ng musk xylene/g, < 2 to 238 ng musk ketone/ g, < 5 to 917 ng HHCB/g, < 5 to 144 ng AHTN/g, and < 10 to 88.0 ng HHCB-lactone/g, on a lipid weight basis. The concentrations of HHCB were higher than the concentrations of other synthetic musks in breast milk samples. The mean concentration of HHCB (220 ng/g, lipid weight) was 5 times greater than the concentrations reported 10 years ago for breast milk samples collected in Germany and Denmark. Maternal age was not correlated with the concentrations of musk xylene, musk ketone, HHCB, or AHTN. There was a trend of decreasing concentrations of musk xylene, musk ketone, HHCB, and AHTN, with the number of children previously breast-fed, although the correlation was not significant. Based on average daily ingestion rate of breast milk, an infant is estimated to ingest 297 +/- 229 ng musk xylene, 780 +/- 805 ng musk ketone, 1830 +/- 1170 ng HHCB, 565 +/- 614 ng AHTN, and 649 +/- 598 ng HHCB-lactone per day. The ingestion rate of synthetic musks by infants in the United States is lower than that estimated for persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs). Based on the residue patterns and accumulation features, it can be concluded that the exposure characteristics for synthetic musks are different from those of POPs, and that the major source of exposure to synthetic musks is probably via dermal absorption or inhalation.

Bioaccumulation of synthetic musks in a marine food chain was investigated by analyzing marine organisms at various trophic levels, including lugworm, clam, crustacean, fish, marine mammal, and bird samples collected from tidal flat and shallow water areas of the Ariake Sea, Japan. Two of the polycyclic musks, HHCB and AHTN, were the dominant compounds found in most of the samples analyzed, whereas nitro musks were not detected in any of the organisms, suggesting greater usage of polycyclic musks relative to the nitro musks in Japan. The highest concentrations of HHCB were detected in clams (258-2730 ng/g lipid wt.), whereas HHCB concentrations in mallard and black-headed gull were low, and comparable with concentrations in fish and crab. These results are in contrast to the bioaccumulation pattern of polychlorinated biphenyls; for which a positive correlation between the concentration and the trophic status of organisms was found. Such a difference in the bioaccumulation is probably due to the metabolism and elimination of HHCB in higher trophic organisms. Temporal trends in concentrations of synthetic musks were examined by analyzing tissues of marine mammals from Japanese coastal waters collected during 1977-2005. HHCB concentrations in marine mammals have shown significant increase since the early 1990s, suggesting a continuous input of this compound into the marine environment. Comparison of the time trend for HHCB with those for PCBs and PBDEs suggested that the rates of increase in HHCB concentrations were higher than the other classes of pollutants. To examine the geographical distribution of HHCB, we have analyzed tissues of fish, marine mammals, and birds collected from several locations. Synthetic musks were not detected in a sperm whale (pelagic species) from Japanese coastal water and in eggs of south polar skua from Antarctica. While the number of samples analyzed is limited, these results imply a lack of long-range transportation potential of synthetic musks in the environment.

A mouse model combining liver-specific deletion with global suppression of the NADPH-cytochrome P450 reductase gene (Cpr) has been developed and characterized. These mice (designated "Cpr-low and liver-Cpr-null" or CL-LCN) retain the respective phenotypes of the previously reported Cpr-low (CL) and liver-Cpr-null (LCN) mouse strains, but hepatic deletion of the Cpr gene occurs at an earlier age in the CL-LCN mouse than in the LCN mouse. Residual hepatic microsomal CPR activities are very low in both CL-LCN and LCN mice (at 1.5 and 2.5% of wild-type levels, respectively). The utility of CL-LCN mice for in vivo drug metabolism studies was explored using the cytochrome P450 (P450) prodrug cyclophosphamide (CPA). After i.p. injection of CPA at 100 mg/kg, the t1/2 and the area under the concentration-time curve for plasma CPA were significantly increased in mice deficient in liver CPR compared with wild-type controls, indicating a lower rate of metabolism, with the effects greater in CL-LCN mice than in LCN mice. Correspondingly, substantial decreases in Cmax, and increases in Tmax, and t1/2, of 4-hydroxycyclophosphamide (4-OH-CPA) formation were observed in both LCN and CL-LCN mice relative to wild-type controls. In contrast, CPA and 4-OH-CPA pharmacokinetic parameters were essentially unchanged in CL mice, relative to wild-type controls. The slower elimination of CPA in CL-LCN mice compared with LCN mice suggests a role for extrahepatic P450 in the in vivo metabolism of CPA and demonstrates the utility of the CL-LCN model in determining the role of extrahepatic P450 enzymes in drug metabolism and chemical toxicity.

Perfluorinated compounds (PFCs) such as perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) have received worldwide attention because of their environmental persistence and widespread distribution. Because of the lack of robust analytical methods and standards to detect all of the PFCs, and their precursors and metabolic intermediates, a mass balance approach involving the determination of total fluorine (TF), followed by fractionation of samples to separately determine inorganic and organic fluorine, is needed. In this study, we have developed a method to determine low microg/L levels of total fluorine (TF) in seawater samples. Further, seawater samples were fractionated into organic and inorganic fractions by extraction with organic solvents, which were then analyzed for TF, extractable organic fluorine (EOF) and inorganic fluorine (IF; i.e., fluoride). Concentrations of known perfluorinated compounds (PFCs) including PFOS and PFOA were also determined in water samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to enable calculation of the fraction of fluorine that is contributed by PFCs to TF. A major proportion of fluorine in seawater was in the form of fluoride (>90% in locations not affected by direct discharges). Nevertheless, within the organofluorine fraction, a major percentage (60-90%) of fluorine still remains unknown in water samples, suggesting the occurrence of other fluorinated acids in addition to known perfluorinated acids. Further studies are needed to identify and quantify the unidentified organofluorines in seawater. Mass balance analysis of total organic fluorine (TOF) and EOF is important, if we are to understand transport and fate of fluorinated compounds in the environment, and if we are to identify the sources of unidentified fluorinated compounds.

Fluoropolymer dispersions are used for coating certain cookware products and food-contact packaging to impart oil and water repellency. Since salts of perfluorooctanoic acid (PFOA) are used as a processing aid in the manufacture of many fluoropolymers, it is necessary to determine if these compounds are still present as residuals after the process used to coat nonstick cookware or packaging, and could be released during typical cooking conditions. In this study, we identified and measured perfluoroalkyl carboxylates (PFCAs), particularly PFOA, and fluorotelomer alcohols (FTOHs; 6:2 FTOH and 8:2 FTOH), released from nonstick cookware into the gas phase under normal cooking temperatures (179 to 233 degrees C surface temperature). PFOA was released into the gas phase at 7-337 ng (11-503 pg/cm2) per pan from four brands of nonstick frying pans. 6:2 FTOH and 8:2 FTOH were found in the gas phase of four brands of frying pans, and the sources of FTOHs released from nonstick cookware are under investigation. We observed a significant decrease in gas-phase PFOA following repeated use of one brand of pan, whereas the other brand did not show a significant reduction in PFOA release following multiple uses. PFOA was found at >5 ng during the fourth use of both brands of pans. FTOHs were not found after the second use of either brand of pans. PFOA was found at 5-34 ng in the vapors produced from a prepacked microwave popcorn bag. PFOA was not found in the vapors produced from plain white corn kernels popped in a polypropylene container. 6:2 FTOH and 8:2 FTOH were measured in the vapors produced from one brand of prepacked microwave popcorn at 223 + 37 ng and 258 +/- 36 ng per bag, respectively, but not measured at >20 ng (LOQ) in the other two brands. On the packaging surface of one brand of microwave popcorn several PFCAs, including C5-C12, 6:2 FTOH, and 8:2 FTOH, were found at concentrations in the order of 0.5-6.0 ng/cm2. This study suggests that residual PFOA is not completely removed during the fabrication process of the nonstick coating for cookware. They remain as residuals on the surface and may be off-gassed when heated at normal cooking temperatures.

Atmospheric bulk samples were collected monthly, for one year, from urban, suburban, and rural sites located in the coastal areas of Korea, for the assessment of depositional fluxes and seasonal variations in atmospheric concentrations of PBDEs. Twenty individual PBDE congeners were found in atmospheric samples, and their depositional fluxes varied from 10.1 to 89.0 microg/m2/year. The highest depositional fluxes were found in two urbanized areas, with a strong urban-rural gradient. The relationship between PBDE and particulate depositional fluxes showed significant correlation for all locations, which suggested the association of PBDEs to particulate phase. Deca-BDE (BDE 209) was the predominant congener (>93%) in all deposition samples, which is consistent with a high consumption of deca-BDE for the flame-retardant market in Korea. Seasonal variability was observed in PBDE concentrations for the two urban sites, whereas no seasonal trend was found for either the suburban or the rural location.

Concentrations of polybrominated diphenyl ethers (PBDEs) were determined in sediment and bivalves collected from 25 coastal locations in Korea. Twenty major PBDE congeners were found in all sediment and bivalve samples. SigmaPBDE20 concentrations ranged from 0.45 to 494 ng/g, dry weight (average 27.8 ng/g dry weight) in sediments, and from 0.38 to 9.19 ng/g, wet weight (average 2.94 ng/g wet weight) in bivalves. The highest concentrations were found at locations near industrial complexes and large harbors, suggesting that human activities contribute to PBDEs contamination in Korean coastal environment. PBDE concentrations measured in our study, excluding BDE congener 209 (deca-BDE), in sediment and bivalves were lower than those reported from other countries; whereas BDE 209 concentrations were comparable to or higher than those reported from other countries. The predominant PBDE congener in sediments and bivalves was deca-BDE, which accounted for >90% and >60% of the total PBDE concentrations in sediment and bivalves, respectively. This is consistent with high consumption of deca-BDE for the flame-retardant market in Korea. Significant correlations existed among BDEs 28, 47, 99, 100, 153 and 154 in sediments and bivalves; however, BDEs 183 and 209 showed little correlation compared with less highly brominated congeners.

The total estrogenic activity of the wastewater from a swine farm in Japan was quantitatively characterized, and the compounds responsible for the estrogenic activity were identified and quantified. The wastewater treatment process consisted of a series of an up-flow anaerobic sludge blanket (UASB) and a trickling filter. Samples were collected at each treatment step, and the total estrogenic activity was determined by use of an in vitro gene expression assay (MVLN; MCF-7 human breast cancer cell stably transfected with the pVit-tk-LUC receptor plasmid). Individual estrogenic compounds were identified and quantified using liquid chromatography-mass spectrometry (LC/MS) and liquid chromatography-tandem mass spectrometry (LC/ MS/MS). To further identify the compounds contributing to the estrogenic activity in the wastewater, the sample extracts were fractionated into 12 fractions (fractions 1-12) by HPLC. The rate of removal of estrogenic activity between the effluent and the influent was greater than 97%. The trickling filter removed the majority of the estrogenic activity. The removal rates of specific estrogenic compounds ranged from 44 to 99%. Estrogenic activity was detected mainly in the fractions containing estrone (El), 17beta-estradiol (betaE2), 17alpha-estradiol (alpha E2), estriol (E3), bisphenol A (alphaPA), and equol (EQ0). The ratios of betaE2-EQc (betaE2 equivalents derived from chemical analysis) to betaE2-EQB (betaE2 equivalent derived from bioassay) in the 12 fractions collectively were contributed by El (17-30%), betaE2 (23-30%), acE2 (<1%), E3 (1-2%), BPA (<1%), and EQO (2-3%) in the influent and El (16-37%), PE2 (<1-7%), alphaE2 (<1%), E3 (<1-3%), BPA (<1%), and EQO (<1%) in the effluent. The compounds responsible for most of the estrogenic activity measured in the bioassay were natural estrogens such as El and betaE2.

Perfluorinated chemicals (PFCs) have been used as surfactants in industrial and commercial products for over 50 years. Earlier studies of the geographical distribution of PFCs focused primarily on the Northern Hemisphere, while little attention was paid to the Southern Hemisphere. In this study, livers from eight species of albatrosses, blood from elephant seal, and blood and eggs from penguins and polar skua collected from the Southern Ocean and the Antarctic during 1995-2005 were analyzed for 10 PFCs. In addition, for comparison with the Southern Ocean samples, we analyzed liver, sera, and eggs from two species of albatrosses from Midway Atoll in the North Pacific Ocean. Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) were found in livers of albatrosses from the Southern Ocean. PFOS was the major contaminant, although the concentrations were <5 ng/g, wet wt, in 92% of the albatross livers analyzed. PFOA was detected in 30% of the albatross livers, with a concentration range of <0.6-2.45 ng/g,wet wt. Other PFCs, including long-chain perfluorocarboxylates (PFCAs), were below the limits of quantitation in livers of albatrosses from the Southern Ocean. In liver, sera, and eggs of albatrosses from the North Pacific Ocean, long-chain PFCAs (perfluorononanoate, perfluorodecanoate, perfluoroundecanoate, and perfluorododecanoate) were found at concentrations similar to those of PFOS and PFOA. The mean concentration of PFOS in livers of Laysan albatrosses from the North Pacific Ocean (5.1 ng/g, wet wt) was higher than that in several species of albatrosses from the Southern Ocean (2.2 ng/g, wetwt). Species-specific differences in the concentrations of PFOS were noted among Southern Ocean albatrosses, whereas geographical differences in PFOS concentrations among the Indian Ocean, South Pacific Ocean, and South Atlantic Ocean were insignificant. Concentrations of PFOS and PFOA were, respectively, 2- and 17-fold higher in liver than in sera of Laysan albatrosses. PFOS was found in the blood of elephant seals from Antarctica at concentrations ranging from <0.08 to 3.52 ng/mL. PFOS was found in eggs (2.1-3.1 ng/g) and blood (<0.24-1.4 ng/ mL) of polar skuas but was not detected in penguins from Antarctica. Our study documents the existence of low but detectable levels of PFOS and PFOA in Southern Hemisphere fauna, suggesting distribution of these compounds on a global scale.