Rainbow trout and other fish species can
serve as “sentinel” species for the assessment
of ecological status and the presence
of certain environmental contaminants.
As such, they act as bioindicators
of exposure. This paper presents seminal
data regarding dose–response and toxicokinetics
of trout hemoglobin adduct
formation from exposure to nitro musks
that are frequently used as fragrance
ingredients in formulations of personal-care
products.
Hemoglobin adducts serve as biomarkers
of exposure of the sentinel species, as has
been shown in previous studies of hemoglobin
adducts formed in trout and environmental
carp exposed to musk xylene (MX) and musk ketone (MK). Gas chromatography-electron capture negative ion chemical ionization-mass spectrometry
(GC-NICI-MS) employing selected ion monitoring (SIM) was used to measure 4-amino-MX (4-AMX), 2-amino-MX (2-AMX), and 2-amino-MK (2-AMK)
released by alkaline hydrolysis from the sulfinamide adducts of hemoglobin.
Dose–response and toxicokinetics were investigated using this sensitive method for
the analysis of these metabolites. In the dose–response investigation, the concentrations
of 4-AMX and 2-AMX were observed to pass through a maximum at 0.10
mg/g. In the case of 2-AMK, the adduct concentration was almost the same at
dosages in the range of 0.030–0.10 mg/g. For toxicokinetics, the concentration of
the metabolites in the hemoglobin (Hb) reaches a maximum in the three-day sample
after administration of MX or MK. Further elimination of the metabolites
exhibited kinetics with a presumed exponential decay and a half-life estimated to
be 1–2 days. This suggests that a robust mechanism of elimination of the adducts
exists in fish erythrocytes apparently analogous to that observed in mammals. Two
sick fish were observed to yield 5–24 times the amount of adducts of similarly
exposed fish, suggesting that this elimination mechanism may have been impaired
or lacking in susceptible individual fish. It appears that adducts are destroyed in
times far shorter than the expected life spans of the erythrocytes. This finding may
have implications for the use of Hb biomarkers as integrative measures of exposure
in some contexts. Additional conclusions from these preliminary data include the
additive burden of exposure to multiple compounds and the increased susceptibility
and direct observation of metabolic differences of individual members of the
species completely independent of habitat and feeding habit variations.
Musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobezene, MX) and musk
ketone (1-tert-butyl-3,5-dimethyl-2,6-dinitro-4-acetylbenzene, MK), the most
prevalent synthetic nitro musk compounds, are frequently used as fragrance and
additive materials in personal-care products and perfumed household products.
They are used as a substitute for expensive natural musk, and their estimated
annual production is about 1000 metric tons.1 Due to their persistence in the environment
and high potential for bioaccumulation,2 MX and MK have been detected
as contaminants in aquatic and terrestrial organisms;3,4 human tissues;5–7 North
Sea, river, and freshwater;1,8,9 sewage treatment effluent;10 Norwegian air samples;11
human adipose tissue and breast milk;2,12 developing and adult rats;13 and fish, mussels,
and shrimp.14 Metabolites of MX and MK have been identified and quantified
in samples of river waters, domestic and industrial sewage sludge,9,15 and homogenized
whole fish tissues.16 Some studies have been reported on the ecotoxicity of
MX and its metabolites17,18 and toxic effects in mice.19 Several studies suggest that
MX is not genotoxic.20–22 MX and MK were identified as inducers of toxifying
enzymes, cytochrome P450 1A1, and 1A2 in the rat liver.23
Fish serve as sentinel species for the assessment of ecological health and the presence
of certain environmental contaminants.24 As such, they function as bioindicators
of exposure and thus provide information
on the status of an entire ecosystem
or watershed.25 The sentinel species,
in turn, may have quantifiable biological
responses to the exposure events. Exposure
events consist of contact with agents or
stressors in the environment that may consist
of traditional contaminants such as
pesticides, or may be nontraditional substances
such as certain endocrine disrupting
compounds (EDCs) or pharmaceuticals
and personal-care products (PPCPs).26
Because of their aqueous habitat, fish
experience continuous exposure to contaminants
that perfuse into receiving
waters. In addition, there is the factor of
cumulative risk as a result of exposure to
multiple stressors.27 For example, the protein vitellogenin appears to be a sensitive
indicator in some fish species for the presence of estrogenic EDCs.28,29
Hb adducts have served as suitable biomarkers for exposure to carcinogenic aromatic
amines and nitroarenes. The metabolites of nitro musks or other related
nitroarenes, bound to Hb as biomarkers of exposure, can potentially be used to
integrate continuous exposures over a longer time range (possibly over the lifetime
of red blood cells), and thus may be better suited for risk assessment than quantitation
of urinary metabolites.30,31
Figure 1 - Metabolic pathway of cysteine Hb adduct formation with nitro musk compound using
4-AMX as the example.
Nitroarenes are subject to enzymatic reduction, and their reactive intermediate,
nitrosoarenes, react with the sulfhydryl (SH) functional group of cysteine in Hb to
form an acid/base labile sulfinamide that hydrolyzes to aromatic amines in the presence
of aqueous base.32 This process is shown schematically in Figure 1. The formation
of Hb adducts suggests that DNA adducts may be formed as well.
The authors detected 4-AMX metabolite from carp Hb for the purpose of ecological
assessment of MX exposures.33 In the course of their earlier studies of trout
exposed to MX and MK, a trout Hb adduct of a 4-AMX was found, suggesting that
nitro-reduction of MX may occur in fish34,35 as well as humans.36
In the present work, the 4-AMX, 2-AMX, and 2-AMK metabolites bound to Hb,
formed by enzymatic reduction of MX and MK, were detected and quantified by GC-NICI-MS using SIM. This was found to be an improvement in sensitivity over
earlier methods of analysis.33,37 To the authors’ knowledge, the present investigation
is the first report on dose–response and toxicokinetics of nitro musk Hb
adducts from the sentinel species of rainbow trout exposed to MX and MK and
therefore is the first report on Hb biomarkers of a sentinel species thus exposed.
Experimental
Standards, chemicals, and solvents
Sodium dodecyl sulfate (SDS), sodium hydroxide pellets, and n-hexane
(HPLC-grade) were purchased from Sigma-Aldrich (St. Louis, MO), Fisher
Scientific (Pittsburgh, PA), and J.T. Baker (Phillipsburg, NJ), respectively.
The internal standard (IS), 2,3,4,5,6-pentafluorobenzophenone (purity 99%),
was obtained from Aldrich Chemical Co. (Milwaukee, WI). MX and MK standards
were a gift from the Institute of Food Chemistry, University of
Hohenheim (Stuttgart, Germany). The metabolites 4-AMX, 2-AMX, and 2-AMK were synthesized from MX and MK by Dr. Lantis I. Osemwengie (U.S.
EPA, Las Vegas, NV10). Purities were estimated to be greater than 98%. Tricane
methane sulfonate (MS 222) was obtained from Sigma (St. Louis, MO).
Deionized water was used for all preparations.
Exposure of trout to nitro
musk compounds
Trout exposure experiments were conducted at
the Department of Environmental &
Molecular Toxicology, Oregon State
University (OSU) (Corvallis, OR) for the
sampling periods of 24 hr (1 day), 72 hr (3
days), and 168 hr (7 days). A series of standard
test solutions containing 10, 30, 100, and 300
mg/mL MX or MK were prepared in salmon oil
as the vehicle (pharmaceutical grade, Yukon
Nutritional Co., Lake Wales, FL) for trout
exposure to MX and MK. At the highest
intended concentration, neither the MX nor
the MK dissolved completely in the oil, but
instead formed an emulsion. Well-shaken standard
solutions were injected intraperitoneally
into fish that were anesthetized in an aqueous
MS 222 solution containing 75 mg/L in a 15-L
tank. The anesthetized trout were weighed
before injecting the standard solutions into the fish.
For the dose–response study, 24 trout were exposed to MX or MK solutions, three
trout for each level. For the toxicokinetic investigations, 12 more trout were exposed
to 30 mg/mL MX or MK, six with each standard solution. For control work, nine fish
were exposed to the vehicle (no MX or MK) for the same sampling period. After
exposure, the fish were returned to labeled tanks with circulating water at 13 °C.
Observation of trout during sampling period
In total, 45 trout were exposed to MX or MK solutions and the salmon oil vehicle,
followed by sampling periods of 1 day, 3 days, and 7 days. Following exposure, no
food was given to the fish, which were closely monitored in the labeled circulating
water tank. Two trout exposed to the 30-mg/mL MX solution were observed to be
sick in the tank on day 1 and day 7. One fish exposed to 10 mg/mL MK died on
day 1 and was not included in the study.
Collection of trout blood and separation of Hb
Before drawing the blood samples, the trout were anesthetized with MS 222 (250
mg/L). This concentration of the MS 222 solution was also fatal to the trout. Blood
samples were drawn from the trout into heparinized individual syringes from the
caudal vein and placed into heparinized individual sterile interior Vacutainers
(Becton Dickinson and Co., Franklin Lakes, NJ). After a 1-day exposure, 23 blood
samples were drawn from 23 trout that were exposed to 10, 30, 100, and 300 mg/mL
MX or MK. Three days and seven days after exposure, 12 blood samples, six on
each exposure day, were collected from 12 trout that were exposed to 30-mg/mL
MX or MK solutions. Nine control blood samples were also drawn from nine trout
for 1 day, 3 days, and 7 days after exposure. All blood samples were placed on ice
immediately after collection, and the fish were sacrificed. Erythrocytes or red blood
cells were separated from plasma by centrifuging at 3500 × g for 10 min at 4 °C and
were washed twice with equal volumes of 0.9% saline and recentrifuged. The red
blood cells were lysed by adding two volumes of distilled water. The Hb solutions
were solidified in a freezer at –24 °C. The solid Hb solutions were shipped
overnight from OSU to the National Exposure Research Laboratory, U.S. EPA
(Las Vegas, NV), in an insulated box packed with dry ice. Upon receipt, the water
was eliminated from the solid Hb solutions by a freeze-drying procedure using a
Sentry Microprocessor Controlled Freezemobile and benchtop freeze-dryer (The
VirTis Co., Inc., Gardiner, NY). The dried Hb was then placed in a freezer at –24
°C for subsequent analysis of the nitro musk metabolites.
Liberation of bound amine metabolites from Hb
Ref. 33 reports on the alkaline hydrolysis, extraction, and preconcentration procedures
for liberation of the bound amino metabolites from the carp Hb. The same
procedures were used in this study. The dried extract (about 45 mL) was concentrated
under a stream of nitrogen at 45 °C to a volume of about 50–65 μL, to which
10–100 pg/μL of IS was added. The solution was sealed in GC vials and analyzed
by GC-NICI-MS using SIM.
Two nonhydrolyzed Hb control experiments were also performed to investigate
the possible presence of unbound amino metabolites in the Hb samples. In the
experiments, all chemicals and solvents, except for the NaOH, were added to the
Hb (about 50 mg), and the same extraction and preconcentration procedures
were followed as described in the alkaline hydrolysis work. A laboratory control
experiment was carried out by utilizing the same amounts of solvents, chemicals,
and reagents used for the hydrolysis, except no Hb was used.