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National Institute of Environmental Health Science.

Annual report : National Institute of Environmental Health Sciences (Volume 1983) online

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October 1, 1982 to September 30, 1983



riTLE OF PROJECT (30 characters gr less. Title musf fiton one line between the borders.)

Transport and Metabolism of Phthalate Esters



PRINCIPAL INVESTIGATOR (List other professional personnel on subsequent pages.)

LMB



(Name, title, laboratory, and institute affiliation)

Phillip W. Albro Research Chemist



NIEHS



COOPERATING UNITS (17 any;

C.C. Pack, M.D., LTC, MC, USUHS
Gary Liss, M.D., CDC



LAB/BRANCH

laboratory of Molecular Biophysics



SECTION

Bio-organic Chemistry



INSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MANYEARS:

2.6



PROFESSIONAL:



0.8



OTHER:

1.6



CHECK APPROPRIATE BOX(ES)

□ (a) Human subjects

□ (a1) Minors

□ (a2) Interviews



(b) Human tissues



□ (c) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not excead the space provided.)

The phthalate portion of diethyl hex.yl phthalate (DEHP) does not bind to
macromolecules , but a portion of the ethylhexyl chain does (in vivo ). Phthalate
was found in each of 200 samples of human urine ; ubiquitous exposure was con-
firmed. Metabolism of DEHP involves extracellular, microsomal, cytoplasmic and
mitochondrial enzymes; over 24 metabolites have been identified in the rat.
Rats are a very poor model for man . The enzymes that oxidize monoethylhexyl
phth'alate in rodents differ from those in primates both in positional and in
stereo-specificity . Both the metabolites and the metabolic process itself
may be involved in the biological activity of DEHP.



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Principal Investigator and All Other Personnel Engaged on the Project : (

Phillip W. Albro Research Chemist LEG NIEHS
James R. Mass Research Chemist LEC NIEHS

Kun Chae Chemist LEB NIEHS

PROJECT DESCRIPTION

METHODS EMPLOYED : Isotopic tracers, chromatographic stripping, selective
derivatization, gas chromatography, radio-HPLC, mass spectrometry, standard
enzymology techniques, DNA isolation using hydroxyapatite, enzymatic digestion.

MAJOR FINDINGS AND PROPOSED COURSE : Incorporation of the labeled carbon atom
from di-(2-ethylLl - ^'^CJ-hexyl) phthalate into DNA in rat liver occurs more
extensively in regenerating than in normal liver, and results in labeling of all
four major nucleosides. Similar labeling of the DNA occurs in mouse liver in
vivo . This phenomenon appears to involve metabolic incorporation into normal
precursors rather than covalent binding. In contrast, the diethyl hexyl
phthalate metabolite 2-heptanone, which represents the seven carbon atoms not
including C-1 from ethyl hexanol , appears to bind to DNA spontaneously. This is
obscured in vivo as 2-heptanone is extensively metabolized to 002- Phthalate
has been found in nearly 200 samples of human urine, and no urine has been found
completely free of it. This study is as yet incomplete. Diethylhexyl phthalate,
after hydrolysis by nonspecific lipase to monoethyl hexyl phthalate, is hydro-
xylated at several sites on the ethyl hexyl chain by liver and kidney microsomes i
in the presence of NADPH in vitro . Oxidation beyond this point does not occur
with purified microsomes. Over 24 metabolites of diethylhexyl phthalate have
been identified in rat urine. Relative levels of the ten major metabolites in
urine have been compared for seven animal species and wide species variability
found. Different species have different absorption thresholds below which
diethylhexyl phthalate is not absorbed intact. Future studies should include
in vitro elucidation of the metabolic pathway, which involves extracellular,
microsomal, supernatant and mitochondrial enzymes. The metabolite distribu-
tions should be determined for normal, diabetic, and rhenopathologic humans.
The effect of chronic exposure to phthalate ester on the NAD(P)/NAD(P)H ratios
in liver should be determined, as well as the biological properties of
the major diethylhexyl phthalate metabolites. Similar studies should be
applied to compounds other than diethylhexyl phthalate.

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : Since
diethylhexyl phthalate is a compound to which everyone in any industrilized
country has a chronic, low level exposure, and since it has been shown to be
associated with hepatocarcinoma in both rats and mice as well as producing
testicular atrophy in male rats, the relationship between its biological
activity and its metabolism in various species including man must be determined.
Since the metabolic pathway is uncommonly elaborate, involving metabolic capa-
bilities not previously anticipated, studies of the metabolic machinery, in rat



278



ZOl ES 30020-1 2-LMB



liver cells especially, should spotlight processes applicable to a wide variety
of xenobiotics. Environmental chemicals whose undesirable biological effects
are mediated by their metabolites or by the process of metabolism itself do not
fit easily into current short-term assays for hazard potential unless their
metabolism can be accomplished using a simple post mitochondrial supernate or
bacteria. The metabolism of phthalates is entirely different in bacteria from
in mammals, and is merely begun by the enzymes present in a post mitochondrial
supernatant. Studies using these compounds as models will generally increase
our ability to both predict and test for health hazards associated with environ-
mental chemicals having highly complex catabolic pathways.

PUBLICATIONS

Albro, P.W., Corbett, J.T., Schroader, J.L., Jordan, S., and Matthews, H.B.:
Pharmacokinetics, interactions with macromolecules, and species differences in
metabolism of DEHP. Environ. Health Persp . 45: 19-25, 1982.

Peck, C.C, and Albro, P.W.: The toxic potential of the plasticizer di-2-
ethylhexyl phthalate in the context of its disposition and metabolism in
primates and man. Environ. Health Persp . 45: 11-17, 1982.

Albro, P.W., Jordan, S.T., Schroeder, J.L., and Corbett, J.T.: Chromatographic
separation and quantitative determination of the metabolites of di-(2-
ethylhexyl) phthalate from urine of laboratory animals. J. Chromatogr . 244:
65-79, 1982.

Albro, P.W., Corbett, J.T., Schroeder, J.L., and Jordan, S.T.: Incorporation of
radioactivity from labeled di-(2-ethylhexyl ) phthalate (DEHP) into DNA of rat
liver in vivi . Chem.-Biol. Interact ., in press.

Albro, P.W., Hass, J.R., Peck, C.C, Jordan, S.T., Corbett, J.T., and Schroeder,
J.: Applications of isotope differentiation for metabolic studies with di-
(2-ethylhexyl) phthalate. J. Environ. Sci. Health , B17; 701-714, 1982.

Sherman, L., Thompson, K., O'Kell, R.T., Albro, P., and Inkster, M.: Phthalate
levels in microwave thawed fresh frozen plasma. Transfusion , in press.



279



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



ZOl ES 30034-08 LMB



PERIOD COVERED

October 1, 1982 to September 30, 1983



TITLE OF PROJECT (30 characters or leas. Title must fit on one line between the borders.)

Chemistry of Aromatic Compounds and Their Environmental Transformation Products



PRINCIPAL INVESTIGATOR (List other professional personnel on subsequent pages.)
(Name, title, laboratory, and institute affiliation)

Louis A. Levy Research Chemist LMB



NIEHS



COOPERATING UNITS (if any)



LAB/BRANCH

Laboratory of Molecular Biophysics



SECTION

Bio-Organic Chemistry



INSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MANYEARS:
2.0



PROFESSIONAL;

2.0



0.0



CHECK APPROPRIATE-BOX(ES)

□ (a) Human subjects

□ (al) Minors

□ (a2) Interviews



□ (b) Human tissues



C^ (c) Neither



SUMMARY OF WORK (Vse standard unreduced type. Do not exceed the space provided.)



The development of rational synthetic routes to polynuclear aromatic hydrocarbons

and their metabol ites have been investigated. Models appropriate to the study of

the chemical, physical and spectroscopic properties of these classes of compounds
as potential persistent environmental agents have been prepared.



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Principal Investigator and All Other Personnel Engaged on the Project :

Louis A. Levy Research Chemist LMB NIEHS

A.R.K. Murthy Visiting Fellow LMB NIEHS

PROJECT DESCRIPTION

METHODS EMPLOYED : Synthetic techniques, organometall ic reagents and catalysis,
high pressure reactions, photochemical processes, functional group transforma-
tions. Mass spectroscopy, nuclear magnetic resonance spectroscopy, other
spectroscopy methods (IR, UV), chromatography (column, glc, hplc).

MAJOR FINDINGS AND PROPOSED COURSE : The new methodology developed for the
synthesis of benzanthracenes has been extented into a new synthesis of 5-
substituted chrysenes, the most biologically active of this class of polynuclear
aromatic hydrocarbons. The stereochemical phenomena associated with some of the
complex benzanthracenes have provided information about their conformations and
the possibility of obtaining pure enantiomers from a racemic mixture of poly-
nuclear aromatic hydrocarbons. Benzanthracenes with a functional group at C-12
have been prepared and the opportunity provided by the presence of this reactive
moiety has been exploited to synthesize a number of benzanthracenes substituted
at C-12 with oxygenated and alkylated groups. Some known and hypothetical
oxygenated metabolites of substituted benzanthracenes have been synthesized.
Several unknown or extremely rare nitrogen substituted benzanthracenes and
benzanthracene derivatives have been prepared.

It is proposed to continue the development and refinement of new synthetic
approaches to polynuclear aromatic hydrocarbons and their metabolites such
as arene oxides and hydroxylated derivatives.

SIGNIFICANCE TO BIO-MEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : Poly-
nuclear aromatic hydrocarbons are recognized to be of major environmental
importance due to their widespread ecological distribution and, in some cases,
their concentration in the food chain. The availability of the unique isomers
of these classes of compounds and their probable metabolites would permit
critical biological and toxicological studies. Synthetic availability of
appropriate model compounds allows further examination of the mechanisms of
their biological activity.

PUBLICATIONS

Levy, L.A. and Kumar, S.: Synthesis of methyl substituted benzanthracenes
and benzanthracene derivatives. Tetrahedron Letters . 24: 1217-1221, 1983.

Levy, L.A.: Synthesis of 2,3,6,7-tetrasubstituted naphthalenes; 2,3,6,7-
tetrachloronaphthalene. Synthetic Comm . (in press).

13 13
Cox, R.H. and Levy, L.A.: "^C - C Spin coupling constants in tetra-

hydronaphthalene, hexahydrobenzanthracene and benzo(a)pyrene derivatives.

Organic Magnetic Resonance. 21: 173-176, 1983.



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Mass, J.R., Bobenreith, M.J. and Levy, L.A.: A study of the electron impact
induced retro diels-alder process with selected 5, 6, 6a, 7, 12, 12a
hexahydrobenzanthracenes. Or£. Mass Spect . (in press).



282



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



ZOl ES 30050-07 LMB



PERIOD COVERED

October 1, 1982 to September 30, 1983



TITLE OF PROJECT (30 characters or less. Title must fit on one line between the borders.)

Chemical and Enzymatic Conjugation of Glutathione with Epoxides



PRINCIPAL INVESTIGATOR (List other professional personnel on subsequent pages.)
(Same, title, laboratory . and institute affiliation)

Oscar Hernandez Senior Staff Fellow



LMB



NIEHS



COOPERATING UNITS (if any)



Marine Pharmacology Section, LP, NIEHS and TRIP,



LAB/BRANCH

Laboratory of Molecular Biophysics



SECTION

Blo-Orqanic Chemistry



INSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MANYEARS:



2.2



PROFESSIONAL:



1.2



1.0



CHECK APPHOPRIATE'BOX{ES)

□ (a) Human subjects
O (a1) Minors
O (a2) Interviews



(b) Human tissues



m (c) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the ipace provided.)



The chemical conjugation of specific benzo[a]pyrene oxides and other epoxides
to glutathione , enzymatically and non-enzymatically, is being investigated
using nuclear magnetic resonance ( NMR ) spectroscopy , chemical synthesis , and
high pressure liquid chromatography ( HPLC ). The regiospecificity and stereo-
specificity of the conjugation reaction is being determined. Procedures for



the synthesis of thioether metabolites of various epoxides have been modified
to allow large scale preparations. The stereochemistry of the reaction of
glutathione with styrene oxide catalyzed by purified rat liver glutathione
transferases has been established, and an active site geometry has been proposed
to account for the stereoselectivity observed in enzyme mediated reactions.



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LMB


NIEHS


LP


NIEHS


LMB


NIEHS



ZOl ES 30050-07 LMB

Principal Investigator and All Other Personnel Engaged on the Project :

Oscar Hernandez Senior Staff Fellow
Jack Bend Research Pharmacologist

M.B. Gopinathan Visiting Fellow

PROJECT DESCRIPTION

1 3 1
METHODS EMPLOYED : Fourier transform C and H nuclear magnetic resonance

spectroscopy; high pressure liquid chromatography; organic synthesis, in vitro

biological experiments.

MAJOR FINDINGS AND PROPOSED COURSE : The reaction of N-acetyl cysteine methyl
ester with epoxides in methanol solution provides a useful, preparative scale,
procedure for synthesis of mercapturic acids of alkene and arene oxides. By this
approach, diastereomerically pure thioethers of styrene oxide, a-methylstyrene
oxide, 3-methyl styrene oxide, 1 ,2,3,4-tetrahydro-naphthalene 1,2-oxide, naphtha-
lene 1,2-oxide, phenanthrene 9,10-oxide, and pyrene 4,5-oxide have been prepared.

The stereochemistry of the enzyme catalyzed reaction of glutathione with styrene
oxide has been explored with purified rat liver transferases. The regiochemistry
and enantioselectivity were found to be dependent on the enzyme and enantiomer
used as well as substrate concentration. The finding that the enantiomers of
styrene oxide differ in mutagenicity towards Salmonella typhimurium TAIOO stresses
the relevance of stereochemical factors in toxication-detoxication mechanisms. i

The information available on the enzymatic reaction of glutathione with
benzo[a]pyrene 4,5-oxide and styrene oxide has suggested a possible catalytic
arrangement for the active site of the enzyme(s). The epoxide substrate is bound
in a hydrophobic pocket, while glutathione is held in proximity and attack occurs
from only one direction. This geometry is thought responsible for the high
stereoselectivity observed in this transformation.

Proposed course includes elaboration of a synthetic scheme for glutathione conju-
gates similar in approach to the one developed for N-acetyl cysteine derivatives,
and establish the stereochemistry of conjugation for various arene oxides in order
to validate the proposed active site geometry for glutathione transferases.

SIGNIFICANCE TO BIO-MEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : The
mechanistic aspects of the glutathione transferase reaction underscore the
relevance of stereochemical factors in influencing the rate of elimination of
enantiomeric oxides via the glutathione pathway.

PUBLICATIONS

Pagano, D.A. , Yagen, B., Hernandez, 0., Bend, J.R. and Zeiger, E.: Mutagenicity
of (R)- and (S) -styrene 7,8-oxide enantiomers and the intermediary mercapturic
acid metabolites formed from styrene 7,8-oxide. Environ . Mutagen . , 4: 575-584,
1982.



NO.



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



ZOl ES 30051-07 LMB



PERIOD COVERED



October 1, 1982 to September 30, 1983



TITLE OF PROJECT (80 characters or leas. Title must fit on one line between the borders.)

Characterization of Specific Binding Toxic Polyhalogenated Aromatic Hydrocarbons



PRINCIPAL INVESTIGATOR (List other professional personnel on subsequent pages. )

(Name, title, laboratory, and institute affiliation)

James D. McKinney Supervisory Research Chemist



LMB



NIEHS



COOPERATING UNITS (if any)



Chemical Pathology Branch TRTP NIEHS



LAB/BRANCH

Laboratory of Molecular Biophysics



Bio-Organic Chemistry



INSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MANYEARS:

1.0



PROFESSIONAL:



0.5



0.5



CHECK APPROPRIATE 'BOX(ES)

□ (a) Human subjects □ (b) Human tissues

□ (a1) Minors

□ (a2) Interviews



ED (c) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)

The guinea pig was used as an extremely sensitive animal model to investigate
structure-toxicity relationships i n polyhalogenated aromatic hydrocarbons .



Results differ from structure activity relationships associated with the dioxin
receptor. A protein binding model is being developed which we hope will show a
better correlation with the observed structure-toxicity results.



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LMB


NIEHS


LMB


NIEHS


TRIP


NIEHS



ZOl ES 30051-07 LMB

Principal Investigator and All Other Personnel Engaged on the Project :

James D. McKinney Supervisory Research Chemist

P.W. Albro Research Chemist

Kun Chae Research Chemist

E.E. McConnell Veterinary Pathologist

PROJECT DESCRIPTION

METHODS EMPLOYED : Synthetic methods along with X-ray crystallography and other
methods for measuring physical/chemical properties with associated equipment and
techniques were used primarily in this phase of the work. Variable temperature
high resolution multi tjiucleignuclear magnetic resonance (NMR) spectroscopy, using
specifically labeled ( C, F, H, etc.) compounds where possible for studying
complex molecular interactions, is also used when solubility is not a problem.
Isolation and characterization of specific binding site(s) in body tissue and
fluid using standard biochemical methods, and modeling of receptor proteins as
possible and required. Computational methods are also being used to aid our
ability to analyze, interpret and rationalize experimental observations and
permit the ability to predict the potential of other compounds to behave
similarly.



i



MAJOR FINDINGS AND PROPOSED COURSE : The guinea pig was used as an extremely
sensitive animal model to investigate structure-toxicity relationships in poly-
halogenated aromatic hydrocarbons. Lethality in guinea pigs appears to be
associated with toxic planar (or coplanar) compounds with potentiation possible
by certain favorably substituted halogenated bi phenyls. This is consistent with
quantum chemical calculations which aid in estimating populations of coplanar
conformers in the halogenated bi phenyls. This differs from the structure-
activity relationship associated with the dioxin receptor. We seek a model based
on polarizabil ities that will account for the observed differences.

A protein binding model is being developed which we hope will show a better
correlation with the observed structure-toxicity results. The exact nature of
this binding is being studied using X-ray crystal lographic, molecular graphic
and theoretical chemistry approaches. Other work will attempt to show the
relationship of such binding to the mechanism of dioxin and related compound
toxicity,

SIGNIFICANCE TO BIO-MEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : There
is increasing evidence that certain highly toxic halogenated hydrocarbons may
have specific binding receptors in biological systems which differ quantitatively
in their ability to bind both halogenated and non-halogenated planar molecules.
Binding propensity and toxicity may be correctable. An understanding of the
specific molecular level interactions involved in binding may permit one to
predict, prevent, or reverse them.



PUBLICATIONS

McKinney, J.D., Gottschalk, K.E. and Pedersen, L. A Theoretical Investigation

of the Conformation of Polychlorinated Biphenyls. J^. Mol . Struct , (in press),

' 286 "



t



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



ZOl ES 30054-06 LMB



PERIOD COVERED



October 1, 1982 to September 30, 1983



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)

Mass Spectrometry Studies on Low Volatility Samples



PRINCIPAL INVESTIGATOR (List other professional personnel on subsequent pages.)
(Name, title, laboratory, and institute affiliation)

J. Ronald Hass Research Chemist LMB



NIEHS



COOPERATING UNITS (if any)



LAB/BRANCH

Laboratory of Molecular Biophysics



SECTION

Analytical Chemistry



INSTITUTE AND LOCATION

lEHS, NIH, Research Triangle Park, North Carolina 27709



TOTAL MANYEARS;



PROFESSIONAL:



1.6



1.2



.4



CHECK APPROPRIATE. BOX(ES)

□ (a) Human subjects iZH (b) Human tissues

□ (a1) Minors

□ (a2) interviews



E] (c) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)



The optics of a double-focusing mass spectrometer were upgraded to increase
mass range and scan rate. Focusing optics were added to a secondary ion mass
spectrometer source. A focused liquid metal ion source was designed. A



number of compounds have been studied by particle beam ionization techniques.



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Principal Investigator and All Other Personnel Engaged on the Project :

J. Ronald Hass Research Chemist LMB NIEHS

R. Stoll Visiting Fellow LMB NIEHS

PROJECT DESCRIPTION

METHODS EMPLOYED : Particle beam ionization mass spectrometry (fast atom
bombardment, secondary ion mass spectrometry, focused liquid metal ion
source); linked B/E scans.

The VG 7070 F mass spectrometer was upgraded by replacement of the magnet,
flight tube and collector housing to bring the instrument to the 70 E
specification. This increases the mass range by a factor of four (to 2800
daltons at 4000 V beam energy) and the maximum scan rate by a factor of
seven to 0.1 seconds/decade (in mass). The fast atom bombardment source
on this instrument is now in routine use in support of IRP research projects.

A secondary ion mass spectrometry (SIMS) type source has been constructed
for the ZAB/2F spectrometer. This source was found to give comparable per-
formance to the commercial version. The incorporation of elementary focusing
optics on the primary ion beam permitted a reduction of beam intensity by a
factor of ten for the same secondary ion yields. As a consequence, sample
depletion was similarily reduced. The overall improvement in sensitivity
for polypeptides was ca. ten-fold.

Particle beam ionization techniques have been applied to a number of
biological molecules. Bombesin and related polypeptides are readily analyzed
by means of these techniques. The structure of the sulfate addition products
of aflatoxins were determined with the assistance of fast atom bombardment
(FAB). The FAB technique was found to permit the measurement of the mass
spectrum of polynucleotides with as many as four nucleic acids. By combina-
tion with MS/MS techniques, the information relating to sequence could be
obtained. We are currently developing a number of collaborative projects
to apply particle beam techniques to specific research problems within the
IRP.

A focused primary ion source with potential beam diameter of 0.5 micron or
less has been designed and is under construction. After evaluation and any
indicated development, this source will be used to begin a new project in
microchemical morphology in environmental/biological specimens.

SIGNIFICANCE TO BIO-MEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : Finding
mass spectrometric methods for the analysis of small polynucleotide-carcinogen
adducts and other small molecule/biopolymer adducts will permit a rapid method
for identification of these compounds, permitting studies of the effects of
neighboring groups upon the reactivity of the site attacked. Such studies
should illuminate the micro-environments important in environmental chemical
carcinogenesis.



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PUBLICATIONS

Walther, H.J., Parker, C.E., Harvan, D.J., Voyksner, R.D., Hernandez, 0.,
Hagler, W.M., Hamilton, P.B., and Hass, J.R.: Analysis of aflatoxins and



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1983) → online text (page 24 of 89)