106
L. Hovander et al.
MeO-PCBs were prepared as described elsewhere (Bergman et al.
1995). 1,2,3,5-Tetrachlorobenzene, 1,2,3,4-tetrachlorobenzene, penta-
chlorobenzene, 2,3,5,6-tetrachloroanisole, 2,3,6-trichloroanisole, 2,3-
dichloroanisole, 2,3-trichloroanisole, 3-methyl butyl nitrite, and N-
chlorosuccinimide (NCS) were obtained from Aldrich (Gillingham,
UK); 1,2,4,5-tetrachlorobenzene and Tin(II) chloride dihydrate were
purchased from Merck (Darmstadt, Germany). 2,3,4-Trichloroaniline
was obtained from Fluka AG; 3,5-dichloroaniline and 2,6-dichloro-4-
nitroanisole were obtained from Acros (Ceel, Belgium); and 1,2-
dichlorobenzene was obtained from Merck-Schuchardt; and copper
bronze from Carlfors bruk (Huskvarna, Sweden). Silica gel chroma-
trations of OH-PCBs in polar bears from the Canada and
Norway were found to be 15 and 30 g/g LW (Sandau 2000).
A summary of OH-PCBs can be found in a review by Letcher
et al. (2000).
Exposure to phenolic compounds has been discussed as a
contributing factor in the disruption of endocrine-related pro-
cesses in wildlife and humans. So-called xenoestrogens are
exemplified by diethylstilbestrol and certain OH-PCBs and
alkylphenols (Toppari et al. 1996). OH-PCB congeners without
chlorine atoms in the hydroxylated phenyl ring and at least one
ortho-chlorine in the other phenyl ring have been reported to
have estrogenic activity in in vitro assays, and in a few cases
also in vivo (Letcher et al. 2000). Phenolic compounds, partic-
ularly halogenated phenolic compounds, have been shown to
interfere with the transport of thyroid hormones by competing
for binding with transthyretin (TTR) (van den Berg 1990).
Certain OH-PCB congeners have up to 10 times the binding
affinity for TTR relative to the endogenous and major thyroid
hormone in blood, thyroxine (T4) (Brouwer et al. 1998; Lans
et al. 1993). The affinity of OH-PCBs for TTR is the likely
mechanism of the highly selective retention of OH-PCBs in the
blood of humans and wildlife (Bergman et al. 1994).
The aim of the present study was to identify additional
OH-PCBs and other PHCs present in human blood, as deter-
mined in a pooled plasma sample from Swedish blood donors.
The identification of the detected PHCs was limited by com-
parisons to the authentic reference standards available from
commercial sources or via chemical synthesis on site. To
achieve these goals, a large number of authentic reference
standards was synthesized and characterized. The methods of
chemical synthesis and extraction of OH-PCBs and PHCs from
plasma is described. The identification of PHCs present in biota
is a first and necessary step to improve the assessment of risk
from exposure to these types of compounds.
˚
tography was performed on Matrex Silica 60 A (Millipore, 0.035–
0.070-mm particles) (Molsheim, France). Silica gel columns (24 cm ϫ
50 mm ID) were used for the preparative separations of the standards.
Chemicals used for analysis: hexane (Hx) and dichlormethane
(DCM) were of pesticide grade (Fisons, Leicestershire, England).
Methyl tert-butyl ether (MTBE), 2-propanol, and potassium hydroxide
(Eka Nobel AB, Bohus, Sweden), potassium chloride (Merck) and
sulfuric acid (98%, w/w, BDH laboratory Supplies, Poole, England)
were of pro analysis quality. Ethanol (99.5%) was purchased from
Kemetyl (Haninge, Sweden). Diazomethane was synthesized as de-
scribed by Furniss et al. (1989). Silica gel (Ͻ 0.063 mesh), purchased
from Macherey-Nagel (Du¨ren, Germany) was activated by heating it
overnight at 280°C and allowing it to cool to room temperature prior
to use. All glassware was heated at 300°C overnight prior to use.
Synthesis of Authentic Reference Standards
Halogenated Phenols: Several commercially available halogenated
phenols were methylated, purified and checked for their purity. The
methyl ethers of the monocyclic halogenated phenols used as reference
standards are listed in Table 2. In addition to these anisoles, other
halogenated phenols were prepared along with their corresponding
methyl derivatives.
Bromochlorophenols: Six phenols substituted both with bromine and
with chlorine (Table 3) were prepared according to the general pro-
cedure described below. The phenol (0.65 mmol–1.7 mmol) was
dissolved in acetic acid (10 ml) and bromine was added. The reaction
mixture was heated at 80°C for 24 h. The reaction was stopped by
addition of sodium bisulfite, and the products formed were extracted
with DCM or Hx. The organic layer was extracted with a sodium
hydrogen carbonate solution, dried over a sodium sulfate layer, and
evaporated. Some of the products were recrystallized in Hx. Further
details of the bromochlorophenols are given in Table 3.
Materials and Methods
Chemicals
4-Hydroxyheptachlorostyrene (Sandau et al. 2000b) was a kind gift
from Dr. Ross Norstrom (Environment Canada, NWRC, Hull, Canada)
and Triclosan (5-chloro-2-[2,4-dichlorophenoxy]phenol) from Ciba-
Geigy, was a kind gift from Margaretha Adolfsson-Erici (ITM, Stock-
holm University, Sweden). 3,5-Dibromo-2-(2,4-dibromophenoxy)phe-
nol was prepared as described elsewhere (Marsh et al. 1998).
2-Chlorophenol, 3-chlorophenol, 2,4-dichlorophenol, 2,5-dichlorophe-
nol, 3,4-dichlorophenol, 2,3,5-trichlorophenol, 2,3,6-trichlorophenol,
2,4,6-trichlorophenol, 3,4,5-trichlorophenol, 2,3,4,5-tetrachlorophe-
nol, 2,3,4,6-tetrachlorophenol, 2,4,6-tribromophenol were from Fluka
AG (Buchs, Switzerland). 4-Chlorophenol and pentachlorophenol
were purchased from Kebo (Stockholm, Sweden), 2,3-dichlorophenol
from Aldrich-Europe (Beerse, Belgium), 3,5-dichlorophenol from
Merck (Mu¨nchen, Germany), and 2,3,4-trichlorophenol and 2,3,5,6-
tetrachlorophenol were obtained from Janssen Chimica (Beerse, Bel-
gium). 2,6-Dichlorophenol, 2-bromophenol, 3-bromophenol, 4-bromo-
phenol, 2,4-dibromophenol, 2,6-dibromophenol, pentabromophenol,
2-bromo-4-chlorophenol, 4-bromo-2-chlorophenol, and 3,5-dibromo-
salicylic acid were all from Aldrich (Steinheim, Germany). 2,5-Dibro-
mophenol was synthesized as described by Marsh et al. (1999).
The following chemicals were used for synthesis of the 20 methoxy-
polychlorobiphenyls (MeO-PCBs) listed in Table 1, while all other
2,4,5-Tribromophenol and 2,3,4-Tribromophenol: 3-Bromophenol
(5.2 g, 30 mmol) was dissolved in acetic acid (25 ml). Bromine (3.1
ml, 60 mmol) dissolved in acetic acid (20 ml) was added (Hodgson et
al. 1933), and the reaction mixture was stirred at room temperature for
1.5 h. The reaction was stopped by the addition of sodium bisulfite,
and the products were extracted with DCM. The organic layer was
washed with water and dried over sodium sulfate. 2,3,4-Tribromophe-
nol and 2,4,5-tribromophenol were isolated as a mixture (600 mg) after
chromatography of the extracted products on an open silica gel column
with Hx:DCM (2:3) as the mobile phase. The two tribromophenols
(600 mg) were then mixed with sodium hydroxide (3 M, 5 ml), ice (20
g), and acetic anhydride (1.5 ml). The mixture was stirred for 2 min.
The resulting precipitate was isolated and dissolved in DCM. The
products, 2,4,5-tribromophenyl acetate and 2,3,4-tribromophenyl ace-
tate, were separated on an open silica gel column with Hx:DCM (1:1)
as the mobile phase. 2,4,5-Tribromophenyl acetate (344 mg, 0.92
mmol) was dissolved in methanol (10 ml). Potassium hydroxide (1 M,
1 ml) was added and the solution was refluxed for 1 h. The solvent was
removed by distillation, and the residue was dissolved in dilute hy-