Species differences in microsomal oxidation and glucuronidation of 4-ipomeanol: Relationship to target organ toxicity

Article abstract of DOI:10.1124/dmd.116.070003

4-Ipomeanol (IPO) is amodel pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissuemacromolecules and can be trapped in vitro as the NAC/NAL adduct. Pronounced species and tissue differences in IPO toxicity are well documented, as is the enzymological component of phase I bioactivation. However, IPO also undergoes phase II glucuronidation, which may compete with bioactivation in target tissues. To better understand the organ toxicity of IPO, we synthesized IPO-glucuronide and developed a new quantitative mass spectrometry-based assay for IPO glucuronidation. Microsomal rates of glucuronidation and P450-dependent NAC/NAL adduct formation were compared in lung, kidney, and liver microsomes from seven species with different target organ toxicities to IPO. Bioactivation rates were highest in pulmonary and renal microsomes from all animal species (except dog) known to be highly susceptible to the extrahepatic toxicities induced by IPO. In a complementary fashion, pulmonary and renal IPO glucuronidation rates were uniformly low in all experimental animals and primates, but hepatic glucuronidation rates were high, as expected. Therefore, with the exception of the dog, the balance between microsomal NAC/NAL adduct and glucuronide formation correlate well with the risk for IPO-induced pulmonary, renal, and hepatic toxicities across species.

Full text of DOI:10.1124/dmd.116.070003

1521-009X/44/10/1598–1602$25.00
http://dx.doi.org/10.1124/dmd.116.070003
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 44:1598–1602, October 2016
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
Short Communication
Species Differences in Microsomal Oxidation and Glucuronidation of
4-Ipomeanol: Relationship to Target Organ Toxicity
Received February 11, 2016; accepted July 22, 2016
ABSTRACT
4-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-
mediated metabolism to reactive electrophilic intermediates that bind
to tissue macromolecules and can be trapped in vitro as the NAC/NAL
and liver microsomes from seven species with different target
organ toxicities to IPO. Bioactivation rates were highest in
pulmonary and renal microsomes from all animal species (except
adduct. Pronounced species and tissue differences in IPO toxicity are dog) known to be highly susceptible to the extrahepatic toxicities
well documented, as is the enzymological component of phase I
bioactivation. However, IPO also undergoes phase II glucuronidation,
which may compete with bioactivation in target tissues. To better
understand the organ toxicity of IPO, we synthesized IPO-glucuronide
and developed a new quantitative mass spectrometry–based assay for
induced by IPO. In a complementary fashion, pulmonary and renal
IPO glucuronidation rates were uniformly low in all experimental
animals and primates, but hepatic glucuronidation rates were
high, as expected. Therefore, with the exception of the dog, the
balance between microsomal NAC/NAL adduct and glucuronide
IPO glucuronidation. Microsomal rates of glucuronidation and P450- formation correlate well with the risk for IPO-induced pulmonary,
dependent NAC/NAL adduct formation were compared in lung, kidney,
renal, and hepatic toxicities across species.
Introduction
phase II glucuronidation (Fig. 1), at least in rats, as assessed from urine
analysis (Statham et al., 1982). Therefore, to better understand the
species-selective and organ-selective toxicity of IPO, we have developed
a microsomal glucuronidation assay for the protoxin and use it here,
together with the previously described assay for trapping the enedial, to
contrast lung, liver, and kidney microsomal bioactivation and de-
toxification of IPO across a range of toxicity-susceptible species, includ-
ing humans.
4-Ipomeanol [IPO; 1-(3-furyl)-4-hydroxy-1-pentanone] is a furano-
terpenoid toxin produced by moldy sweet potatoes (Ipomoea batatas),
which, if ingested by livestock, can cause interstitial pneumonia, which
is characterized by pulmonary edema and emphysema (Wilson et al.,
1971). Beyond the poisoning of livestock, IPO is a pulmonary toxin in
many laboratory animal species, which bioactivate this protoxin to
a reactive intermediate that binds covalently to macromolecules pre-
dominantly in the lung (Boyd, 1976; Gram, 1989). In certain circum-
stances, IPO is also a renal toxin, at least in male mice (Dutcher and
Boyd, 1979). IPO has also been administered to humans with the intent
of treating cancer patients in whom non–small cell lung cancer was
Materials and Methods
Chemicals. IPO was a gift from the National Cancer Institute (Bethesda, MD).
The IPO-NAC/NAL adduct was prepared as described previously (Baer et al.,
diagnosed (Christian et al., 1989). However, this proved to be un- 2005). NAC, NAL, CHAPS, Escherichia coli b-glucuronidase, silver carbonate,
sodium methoxide, 4-methylumbelliferone glucuronide (4-MBG), Tris, magne-
sium chloride, and UDPGA were purchased from Sigma-Aldrich (St. Louis, MO).
Acetobromo-a-D-glucuronic acid methyl ester was purchased from Toronto
Research Chemicals (Toronto, Canada).
successful; instead, a dose-limiting liver toxicity was observed in these
patients (Rowinsky et al., 1993). Therefore, IPO-mediated toxicity
exhibits species, gender, and organ selectivity.
The bioactivation of IPO may proceed via P450-mediated oxidation
of the furan ring to an enedial intermediate that can be trapped by
reaction with nitrogen and sulfur nucleophiles that generate a stable
S-substituted pyrrole that has been fully characterized (Baer et al., 2005).
The extrahepatic P450 enzyme CYP4B1 exhibits high activity for IPO
bioactivation, and indeed CYP4B1 is a prominent P450 enzyme that is
present in the lung of animal species that are susceptible to pulmonary
toxicity (Baer and Rettie, 2006). However, IPO also undergoes extensive
Biologic Materials. Pooled microsomes from male Beagle dog, male Sprague-
Dawley rat, male cynomolgus monkey, and pooled mixed-gender human tissue
microsomes were purchased from XenoTech LLC (Lenexa, KS). New Zealand
White male rabbit tissues were obtained from R&R Research (Stanwood, WA).
C57BL/6 male mice were obtained from The Jackson Laboratory (Bar Harbor,
ME). Bovine tissue was from Pel-Freez Biologicals (Rogers, AR). Rabbit, mouse,
and bovine liver, kidney, and lung microsomes were prepared by differential
centrifugation, according to standard protocols, from tissue pooled from one to
five animals.
Synthesis of IPO-Glucuronide. In a dark environment, a solution of
acetobromo-a-D-glucuronic acid methyl ester (106 mg, 0.35 mmol) in dry
dichloromethane (0.975 ml) was added dropwise to a suspension of IPO (23 mg,
0.14 mmol), silver carbonate (106 mg, 0.39 mmol), and 4 Å molecular sieves
(40 mg) in dry dichloromethane (2.275 ml) at 240ꢀC. After stirring for 1 hour
This study was supported in part by the National Institutes of Health [grant
R01GM49054] and by the University of Washington School of Pharmacy Brady
Fund for Natural Products.
dx.doi.org/10.1124/dmd.116.070003.
ABBREVIATIONS: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate; GST, glutathione-S-transferase; HRMS, high-
resolution mass spectrometry; IPO, 4-ipomeanol; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 4-MBG, 4-methylumbelliferone
glucuronide; NAC, N-acetyl cysteine; NAL N-acetyl lysine; P450, cytochrome P450; UDPGA, uridine 59-diphosphoglucuronic acid.
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Species Differences in 4-Ipomeanol Microsomal Metabolism
1599
Fig. 1. Schematic illustrating bioactivation versus
inactivation routes for 4-IPO after either P450- or
uridine 59-diphospho-glucuronosyltransferase-mediated
metabolism.
at 240ꢀC, the ice bath was removed, and the suspension was stirred for 24 hour
The suspension was subsequently filtered through a pad of silica and washed
with dichloromethane. The crude mixture was evaporated to dryness and
dissolved in 3.0 ml of dry methanol followed by the addition of 0.3 ml of
a 0.5 M solution of sodium methoxide in methanol. The mixture was stirred for
3 hours at room temperature and was subsequently concentrated in vacuo. Next,
the crude residue was purified by silica gel chromatography (20% methanol/
dichloromethane1% formic acid) to afford 9.6 mg of a pale yellow oil (20% yield
over two steps). Further purification by semi-preparative chromatography
afforded 98.5% pure IPO-glucuronide as a mixture of diastereomers.
centrifuged for 10 minutes at 4000 rpm to remove protein. Samples were then
analyzed for IPO-glucuronide as described above.
Assay for Bioactivation of IPO. In vitro bioactivation of IPO was measured
after a 20-minute incubation of microsomal preparations (0.1 mg of protein, 250-ml
final volume) in triplicate with IPO (50 mM), with mass spectrometry analysis of the
NAC/NAL adduct, as described previously (Parkinson et al., 2013). Fragmentation of
the NAC/NAL adduct was monitored at m/z 452 → 353 by LC-MS/MS on
a Micromass Quattro II Tandem Quadrupole Mass Spectrometer (Waters, Milford,
MA) operating in electrospray-positive mode coupled to a LC system (Shimadzu,
Kyoto, Japan) using a Thermo Hypersil gold 100 Â 2.1 mm column with a particle
size of 3 mm. Samples were eluted with 10 mM formic acid in water (aqueous phase,
solvent A) and 10 mM formic acid in methanol (organic phase, solvent B) at a flow
rate of 300 ml/minute. The initial conditions were 70% solvent A and 30% solvent B,
which increased to 100% solvent B between 2 and 4 minutes and remained at 100%
solvent B until the end of the run at 5 minutes. Under these conditions, the
NAC/NAL-IPO adduct was eluted at 4.7 minutes at a cone voltage of 65 V and
collision energy of 30 V, and the internal standard, furafylline (m/z 261.3 → 80.5),
was eluted at 4.55 minutes, at a cone voltage of 35 V and collision energy of 25 V.
NMR Spectroscopy and High-Resolution Mass Spectrometry of the
Synthesized IPO Glucuronide. 1H NMR spectra were obtained on an Agilent
500 MHz NMR spectrometer. Proton chemical shifts (d) are reported in ppm
from an internal standard of methanol (3.31). Coupling constants (J) are
reported in Hertz, and proton chemical data are reported as follows (where
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad,
ovlp = overlapping): 1H NMR (500 MHz, CD3OD), d (in ppm): 1.22
(d, J = 6.4 Hz, 3H), 1.28 (d, J = 5.87 Hz, 3H), 1.81–1.94 (m, 4H), 2.85–2.97
(m, 1H), 3.00 (t, J = 7.6 Hz, 2H), 3.10 (t, J = 7.6 Hz, 1H) 3.13–3.18 (m, 1H),
3.23 (t, J = 7.6 Hz, 1H), 3.41 (d, J = 8.8 Hz, 2H), 3.44–3.50 (m, 2H), 3.64
(d, J = 8.31 Hz, 2H), 3.84–4.01 (m, 2H), 4.36 (dd, J = 14.2, 7.83 Hz, 2H), 6.77
(s, 2H), 7.56 (s, 1H), 7.58 (s, 1H), 8.38 (s, 1H), and 8.43 (s, 1H). High-
resolution mass spectrometry (HRMS) was acquired on a Thermo Fisher LTQ
Orbitrap equipped with an electrospray ionization probe. HRMS (ESI) was
calculated for C15H19O9 [M-H]2 at 343.1024 (found 343.1033, error 2.6 ppm).
Enzymatic Assay for Glucuronidation of IPO. IPO glucuronidation was
measured following incubation of microsomal preparations (0.1 mg protein) in
triplicate with IPO (50 mM), Tris buffer (100 mM, pH 8.4), UDPGA (5 mM),
CHAPS (0.5 mg/ml), and magnesium chloride (10 mM). After 60 minutes at 37ꢀC
in a shaking water bath, 250-ml reactions were terminated by the addition of an
equal volume of ice-cold methanol containing the internal standard, 4-MBG, and
centrifuged for 10 minutes at 4000 rpm to remove protein.
Mass Spectrometry Analysis of IPO-Glucuronide. Formation of IPO-
glucuronide was measured by liquid chromatography-tandem mass spectrometry
(LC-MS/MS) using the same instrument, chromatographic column, mobile
phases, and gradient elution described earlier for analysis of the NAC/NAL
adduct (Parkinson et al., 2013). Under these conditions, IPO-glucuronide,
monitored by fragmentation of the parent sodiated glucuronide to the aglycone
fragment m/z 367 → 191, with a cone voltage of 35 V and collision energy of
22 V, eluted at 4.77 minutes. The internal standard, 4-MBG, eluted at 4.61 minutes
and was monitored by the transition m/z 375 → 199 with a cone voltage of 30 V
and collision energy of 15 V. Using this assay, the limit of quantitation for IPO-
glucuronide activity was 8 pmol/mg/60 minutes.
Results and Discussion
Study Design. We assessed lung, kidney, and liver microsomal IPO
bioactivation to the NAC/NAL adduct and the competing reaction of IPO-
glucuronidation in a wide variety of species, including common laboratory
animals (mouse and rabbit), human and nonhuman primates, toxicology
species used in preclinical testing of new drug entities (rat and dog) and
livestock that are the target of unintentional poisoning due to the consumption
of moldy sweet potatoes (bovine). A substrate concentration of 50 mM was
chosen for all studies because this plasma concentration, which is relevant to
human liver toxicity at least, was achieved in early human cancer studies
(Rowinsky et al., 1993; Lakhanpal et al., 2001). Extended incubation times,
up to an hour for the glucuronidation assays, were chosen to facilitate direct
comparisons of highly varying levels of ipomeanol glucuronidation across
multiple tissues and multiple animal species.
Validation of IPO-Glucuronide Metabolite. IPO-glucuronide was
synthesized using a standard Koenigs-Knorr reaction between IPO and
acetobromo-a-D-glucuronic acid methyl ester. After deprotection of the ester
groups and column purification, IPO-glucuronide was isolated in high purity.
1H NMR spectra and HRMS data for the synthetic product conform to the
IPO-glucuronide structure. Supplementation of rabbit liver microsomes with
UDPGA and IPO led to the formation of a metabolite at 4.82 minute (Fig.
2A). This peak, which cochromatographed with the synthetic glucuronide
standard, was abolished in the absence of cofactor (Fig. 2B) and when the
sample was pretreated with b-glucuronidase (Fig. 2C).
b-Glucuronidase Treatment of UDPGA-Supplemented Microsomal
Incubations. Rabbit liver uridine 59-diphospho-glucuronosyltransferase incuba-
tions were treated with an equal volume of potassium phosphate buffer (100 mM)
pH 6.0 to adjust the pH of the incubation to ;6.8. Fifty units of b-glucuronidase
was added, and the mixture was incubated for 60 minutes at 37ꢀC in a shaking
water bath in a final reaction volume of 500 ml. Reactions were terminated by the
IPO Glucuronidation in Liver, Kidney, and Lung Tissues. The
highest rates of IPO glucuronidation were found in liver microsomes of
addition of an equal volume of ice-cold methanol containing 4-MBG and all species examined but with high interspecies variability (Fig. 3A).

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Parkinson et al.
Fig. 2. Extracted ion chromatograms (m/z 367 → 191) for
microsomal IPO-glucuronide formation by rabbit liver
microsomes analyzed as detailed in Materials and Methods.
The x-axis and y-axis are time and ion abundance,
respectively. (A) Chromatogram of IPO-glucuronide forma-
tion by rabbit liver microsomes coincubated with UDPGA
and CHAPS. (B) Chromatogram from rabbit liver micro-
somal incubations lacking UDPGA. (C) Chromatogram
obtained after postincubation treatment of complete rabbit
liver incubations with b-glucuronidase.
Hepatic glucuronidation rates were highest in mouse, rat, and rabbit species (Dutcher and Boyd, 1979). IPO bioactivation rates in liver
liver, and much lower in cow, dog, monkey, and human liver. The microsomes from lung toxicity-susceptible species were lower than in
highest rate of extrahepatic glucuronidation occurred in rabbit kidney, the corresponding lung microsomes, again in all cases except the dog.
but this still represented only some 13% of the rate measured in rabbit
IPO has long served as a model compound for studying the role of
liver. Several extrahepatic rates of IPO glucuronidation were close to or reactive intermediates in chemical pneumotoxicity. Studies using
below the limit of quantitation at this substrate concentration. In radiolabeled IPO to monitor covalent binding to microsomes demon-
experimental animals, lung is the principal target organ for toxicity, strated good rank order correlations between the extent of covalent
but the IPO glucuronidation rates are uniformly low relative to hepatic binding and target tissue toxicity, especially across tissues from a given
rates. In male mice, kidney toxicity is also well established, secondary to species (Dutcher and Boyd, 1979; Slaughter et al., 1983). These seminal
the high rates of bioactivation in this organ (Parkinson et al., 2013), observations were consistent with the generation of a highly reactive
where we also find IPO glucuronidation to be negligible.
intermediate of IPO within the tissues where toxicity was manifest. It is
IPO Bioactivation in Liver, Kidney, and Lung Tissues. In contrast now apparent that a major catalyst of IPO bioactivation is the
to pulmonary glucuronidation, high rates of IPO bioactivation were extrahepatic P450 enzyme CYP4B1, whose paralogs across many
measured in lung microsomes from most species, including those known species are present at high concentration in the lung (and male mouse
to be susceptible to pulmonary toxicity (e.g., cow, rat, mouse, and rabbit) kidney), but not in the liver (Baer and Rettie, 2006; Parkinson et al.,
(Fig. 3B). The exception was the beagle dog, where, comparatively, very 2012; Parkinson et al., 2013). Despite these strong associations between
low rates of lung microsomal activity were detected. Human lung P450-mediated bioactivation of IPO by CYP4B1 and target tissue
bioactivation was also minimal, but this is to be expected since native toxicity, and the attractiveness of a tissue-specific form of P450 to
human CYP4B1 is an unstable enzyme with minimal activity toward explain species variation in target organ selectivity of IPO, alternative
IPO (Wiek et al., 2015). Kidney bioactivation of IPO was near baseline pathways for the metabolism of IPO need to be considered (Connelly
in all species examined except male mice, where the rates of NAC/NAL and Bridges, 1986). Whereas substantial progress has been made on
adduct formation were the highest recorded. This is in line with the the detailed enzymology of IPO bioactivation, very little has been
pronounced androgen-driven expression of CYP4B1 in mouse kidney established for routes of detoxification of the parent compound. It is
(Isern and Meseguer, 2003) and the known nephrotoxicity of IPO in this known that IPO glucuronide is a major urinary metabolite of this

Species Differences in 4-Ipomeanol Microsomal Metabolism
1601
A
Fig. 3. Tissue- and species-selective IPO glucuroni-
dation (A) and NAC/NAL formation (B). Metabo-
lites were generated in microsomal incubations
and analyzed by LC-MS/MS as described in the
Materials and Methods section. To better enable
comparisons of reaction velocities across multiple
species and tissue with hugely different IPO
bioactivation and glucuronidation capacities, micro-
somal incubations were conducted for extended
times, up to 60 minutes for IPO glucuronidation.
Note that the y-axis scales for the two panels are very
different. Therefore, for reference and to facilitate
comparisons of tissue rates for the two reactions
from the graph, the lowest quantified rate of
glucuronidation was 9.4 pmol/mg/60 minutes for
rat lung microsomes. The lowest quantified rate of
IPO adduct formation was 22 pmol/mg/20 minutes
for monkey lung microsomes.
B
protoxin in rats (Statham et al., 1982), and one that is likely upregulated male mice is also inefficient in comparison with bioactivation of the toxin
by phenobarbital treatment to the extent that it may reduce pulmonary in this organ.
covalent binding and lethality in this rodent species (Statham and Boyd,
In humans, administration of IPO to lung cancer patients in the 1990s
1982). Therefore, the main goal of this article was to characterize both failed to precipitate the desired lung toxicity (Rowinsky et al., 1993), and
microsomal glucuronidation and oxidative bioactivation of IPO in instead a dose-limiting liver toxicity was observed (Lakhanpal et al.,
toxicity-susceptible species and humans to evaluate the balance between 2001). If the liver toxicity reported in humans was related to P450-
these competing pathways.
mediated metabolism of IPO in that organ, the relative tissue microsomal
The data presented here demonstrate that the in vitro NAC/NAL bioactivation and glucuronidation profiles reported here would also
bioactivation assay is generally predictive of the site of IPO toxicity seem to be predictive for hepatotoxicity. However, this is likely to be
across most species and target organs. The high level of IPO bioactivation overly simplistic, as these microsomal experiments do not take into
in pulmonary microsomes in all species relative to bioactivation in liver account potential metabolic quenching of the reactive species by other
microsomes (with the notable exception of the dog) correlates well with enzymes, such as cytosolic glutathione-S-transferase (GST). Variable
the reported species selectivity regarding pulmonary toxicity. The new GST activity can also be a critical factor in dictating chemically induced
microsomal glucuronidation data strongly complement the bioactivation organ toxicities across species, as exemplified by aflatoxin-induced liver
data by underlining the fact that competing pulmonary glucuronidation is toxicity that is evident in rats, but not mice. In this classic case, the
minimal in all species examined. Moreover, renal glucuronidation in species difference could be attributed to differential GST activities

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Parkinson et al.
Dutcher JS and Boyd MR (1979) Species and strain differences in target organ alkylation and
toxicity by 4-ipomeanol. Predictive value of covalent binding in studies of target organ toxicities
by reactive metabolites. Biochem Pharmacol 28:3367–3372.
Gram TE (1989) Pulmonary toxicity of 4-ipomeanol. Pharmacol Ther 43:291–297.
Isern J and Meseguer A (2003) Hormonal regulation and characterisation of the mouse Cyp4b1
gene 59-flanking region. Biochem Biophys Res Commun 307:139–147.
toward aflatoxin epoxide (Ramsdell and Eaton, 1990). Future studies
that evaluate IPO bioactivation in more complex biologic milieus (e.g.,
S9 fractions) may help to further refine our understanding of species
differences in the target organ toxicity of IPO.
Lakhanpal S, Donehower RC, and Rowinsky EK (2001) Phase II study of 4-ipomeanol, a naturally
occurring alkylating furan, in patients with advanced hepatocellular carcinoma. Invest New
Drugs 19:69–76.
Parkinson OT, Kelly EJ, Bezabih E, Whittington D, and Rettie AE (2012) Bioactivation of
4-Ipomeanol by a CYP4B enzyme in bovine lung and inhibition by HET0016. J Vet Pharmacol
Ther 35:402–405.
Parkinson OT, Liggitt HD, Rettie AE, and Kelly EJ (2013) Generation and characterization of
a Cyp4b1 null mouse and the role of CYP4B1 in the activation and toxicity of Ipomeanol.
Toxicol Sci 134:243–250.
Departments of Medicinal Chemistry
(O.T.P., A.M.T., D.W., A.E.R.) and
Pharmaceutics (E.J.K.), University
of Washington, Seattle, Washington
OLIVER T. PARKINSON
AARON M. TEITELBAUM
DALE WHITTINGTON
EDWARD J. KELLY
ALLAN E. RETTIE
Ramsdell HS and Eaton DL (1990) Mouse liver glutathione S-transferase isoenzyme activity
toward aflatoxin B1-8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide. Toxicol
Appl Pharmacol 105:216–225.
Rowinsky EK, Noe DA, Ettinger DS, Christian MC, Lubejko BG, Fishman EK, Sartorius SE,
Boyd MR, and Donehower RC (1993) Phase I and pharmacological study of the pulmonary
cytotoxin 4-ipomeanol on a single dose schedule in lung cancer patients: hepatotoxicity is dose
limiting in humans. Cancer Res 53:1794–1801.
Slaughter SR, Statham CN, Philpot RM, and Boyd MR (1983) Covalent binding of metabolites of
4-ipomeanol to rabbit pulmonary and hepatic microsomal proteins and to the enzymes of the pul-
monary cytochrome P-450-dependent monooxygenase system. J Pharmacol Exp Ther 224:252–257.
Statham CN and Boyd MR (1982) Effects of phenobarbital and 3-methylcholanthrene on the
in vivo distribution, metabolism and covalent binding of 4-ipomeanol in the rat; implications for
target organ toxicity. Biochem Pharmacol 31:3973–3977.
Authorship Contributions
Participated in research design: Parkinson, Teitelbaum, Kelly, Rettie
Conducted experiments: Parkinson, Teitelbaum, Wittington
Contributed new reagents or analytic tools: Teitelbaum
Wrote or contributed to the writing of the manuscript: Parkinson, Teitelbaum,
Kelly, Rettie
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Address correspondence to: Allan E. Rettie, Box 357610, School of Pharmacy,
University of Washington, Seattle, WA 98195-7610. E-mail: rettie@uw.edu
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