Copyright
National Institute of Environmental Health Science.

Annual report : National Institute of Environmental Health Sciences (Volume 1982) online

. (page 14 of 90)
Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1982) → online text (page 14 of 90)
Font size
QR-code for this ebook


cytes of F] mice or tissue culture cell lines bearing the desired MHC determinants
in haploid fashion, either of which are heterozygous at all MHC loci, are used as
targets. The cells are sequentially treated in suspension at 4°C for 45-60 min
with the proper dilution of two monoclonal antibodies directed against different
alleles of the same MHC locus, one of which is fluorescein- and one rhodamine-
labeled. The cells are then extensively washed and examined in a fluorescent
microscope. 3. Quantitation of Mutation at the MHC Loci : The lymphocytes to be
tested are obtained from normal, untreated mice or from mice exposed to different
doses of various known or suspected mutagens in vivo . Alternatively, lymphocytes
of mice or tissue culture cells are treated in vitro with these mutagens. Follow-
ing the reaction with the fluorophore-labeled monoclonal antibodies, cells are
quantitated for reaction with one, two, or none of the monoclonal antibodies. The
very infrequent occurrence of reaction with one of the antibodies but not the other
is considered to be an indication of a presumed mutation. The spontaneous or back-
ground mutation rate is compared with that in duced by mutagenic treatment. This
screening procedure will, in the future, be greatly simplified by the fluorescence-
activated cell sorter, which has the capacity to screen millions of cells in a
very short period of time for single or dual fluorescence. Furthermore, the pre-
sumed mutant cells can be isolated and further analyzed to determine whether a
heritable mutation has occurred.

MAJOR FINDINGS AND PROPOSED COURSE : Cell lines secreting monoclonal antibodies
against defined determinants of the MHC loci have been obtained and antibody se-
creted into either the tissue culture supernatant or the mouse ascites fluid has
been purified and labeled with fluorophores. Several target cell lines and the F]
generation of several murine inbred lines, which are heterozygous at each MHC locus,
have been obtained. Preliminary experiments to determine the sensitivity of the
fluorescent technique will be performed; and when the detection level is such that
rare mutagenic events can be quantitated, murine lymphocytes or target cell lines
will be treated either in vitro or jn^ vivo^ with a known mutagen, such as ethyl -
nitrosourea (END) or procarbazine, and the induced mutation rate compared to the
spontaneous mutation rate at the MHC locus. This course will be repeated when the
fluorescence-activated cell sorter (FACS) becomes available for use. Cells detec-
ted as mutants by the FACS will be sorted out and grown in tissue culture to de-
termine whether the presumed mutants cells are indeed structurally variant at the
MHC locus due to a heritable mutation aL the DNA level.



165



ZOl ES 65030-01 LBG

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : These studies
are part of the Institute's program to develop and test systems that could be used
to study mutation in mammals in vivo and in vitro using single cells and readily
available somatic tissues such as blood cells. When fully developed this system
could be used to screen chemicals for their mutagenic/carcinogenic activity in a
mammalian system relevant to the human population, and to monitor human populations
for any genetic alterations, possibly as a result of exposure to mutagenic or
carcinogenic chemicals.



166



LABORATORY OF ENVIRONMENTAL BIOPHYSICS



167



LABORATORY OF ENVIRONMENTAL BIOPHYSICS
Summary Statement

The Laboratory of Environmental Biophysics is concerned with two main areas of
research: The biological effects of physical factors present in our environment
(Non-ionizing Radiation, Noise, Light) and the molecular interactions that occur
between environmental agents and their biological targets (Molecular Biophysics).
The physical factors under current investigation include nonionizing radiation
(microwaves), noise (including both auditory and non-auditory effects) and light.
The Molecular Biophysics Program is mainly focused on the use of sophisticated
spectroscopic techniques to monitor the interaction of environmental agents with
nucleic acids, membranes, proteins and microsomal systems. The Laboratory is
organized into three separate Work Groups: Nonionizing Radiation, Noise Bio-
effects, and Molecular Biophysics.

NONIONIZING RADIATION '^

Within the nonionizing radiation program, research is being conducted to: develop
microwave exposure systems for bioeffects research; develop and test techniques
for measuring microwave energy absorption; determine how 2450 MHz microwave radia-
tion interacts with biological systems at the subcellular, cellular, organ and
whole animal level of complexity.

A waveguide system for exposing lobster nerves which allow for intercellular re-
cording of action potentials during irradiation has been developed. This system
has been designed so that accurate measurment of specific absorption rates (SAR's)
can be made and the nerves maintained at a constant temperature during exposure.
A calorimeter system for measuring whole-body SAR's has been assembled and cali-
brated. SAR's in rat pups ranging in weight from 5 grams to 300 grams have been
measured with the system as well as SAR's in Japanese quail eggs. The circularly
polarize waveguide system has been received and is in the process of being assembled,

Research into the biological effects of microwave radiation at the subcellular,
cellular, and organ levels continues to be an important component of the non-
ionizing radiation program. Hepatic lysosomes were exposed to 2.45 GHz at SAR's
of 10, 50, and 100 mW/g for 90 minutes. No effects on lysosomal fragility as
determined by release of lysosomal enzymes, cathepsin D and 3-glucuronidase were
noted. Mitochondria were exposed to 2.45 GHz at an SAR of 100 mW/g. The data
indicate that states II, III, and IV and respiratory control rates were altered.
Microwave radiation at a frequency of 2.45 GHz did not affect either microtubular
protein polymerization or depolymerization when measured using circular dichroism
spectroscopy during exposure to SAR's up to 100 mW/g. The binding of calcium to
erythocyte ghost was not changed by 2.45 GHz microwaves at SAR's up to 200 mW/g as
measured by spectrophotofluorimetry techniques during exposure. Isolated frog
sciatic nerves were exposed to 2.45 GHz microwave radiation, sine-wave modulated
at 8, 16 and 32 Hz. The nerves were exposed to SAR's of 10 and 50 mW/g. A loss
in vitality occurred at 50 mW/g but not at 10 mW/g for all modulation frequencies.
These results suggest that the loss in nerve vitality is nonlinear with respect to
microwave intensity since CW and pulse microwaves produced the effect at an SAR of
10 mW/g. This type of nonlinear behavior would be expected if the neural membrane
is ac';ing as a diode-like detector of the microwave field. Mature spermatocytes
from :urkey semen were exposed for 30 minutes to 2.45 GHz microwaves at SAR's of



169



10 and 50 mW/g. Microwaves did not alter membrane permeability to a vital stain
or the intracellular enzymes LDH and GOT. The in vivo performances of the sperm
following in vitro irradiation was also examineHT The fertility of virgin turkey
hens given a single insemination of microwave exposed sperm was similar to the
nonexposed sperm. Hatchability and embryonic mortality were not different among
the treatment groups. Hematological changes in the turkey poults from eggs
fertilized by exposed sperm were observed. In the group exposed to 50 mW/g a
significant decreases in hematocrit and lower numbers of RBC's were measured at 2
and 4 weeks of age. At 5 and 22 weeks of age the differences had vanished indicat-
ing a compensatory characteristic with increased age.

A significant effort has been made to determine the teratogenic and developmental
effects of 2.45 GHz microwave radiation. Studies examining the effect on brain
development have been completed. Pregnant rats were exposed to 10 mW/cm^ (SAR =
2 mW/g) for 3 hours per day from day 4 of pregnancy and exposure of the offspring
to 10 mW/cm^ was continued through 40 days post partum. No significant effect on
brain development was observed from histological examination. However, when some
of the rat pups were tested in a swim test, the exposed rats has a significantly
shorter swim time to exhaustion than the control rats. Brain development in
Japanese quail embryos exposed to 5 mW/cm^ (SAR =• 4.03 mW/g) 24 hours per day from
day 1 to day 12 of development was also studied.' A slight but statistically
significant retardation was found in the development of the cerebellar cortices at
days, 12, 13 and 14 of development. Some quail were allowed to hatch and were
examined at 8 weeks of age. No significant differences were noted between irradi-
ated and control cerebella at this time. Other Japanese quail eggs were subjected
to 2.45 GHz CW microwave radiation at 5 mW/cm^ (SAR = 4.03 mW/g) during the first
12 days of embryogeny. Following hatching the exposed embryos, as well as non-
exposed controls, were reared to 22 weeks of age. Humoral immune potential, as
indicated by comparable anti-CRBC antibody, IgM and IgG, levels at 0, 4 and 7 days
post- immunization in both exposed and control quail, was not affected significantly.
However, cell mediated immune potential measured by the reaction to introdermal
injection of phytohemagglutinin-P in the wing web, was reduced in the exposed
females, but not in the exposed males. Additionally, total leucocyte numbers and
absolute circulating numbers of lymphocytes, monocytes and heterophils were
increased significantly only in the exposed females. These data show that exposure
of Japanese quail during embryogenesis reduced cell mediated immune potential and
induced a general leucocytosis in females,

NOISE BIOEFFECTS

The Noise Effects Workgroup conducts research to: identify the physiological,
biochemical and structural mechanisms that lead to cellular and neural damage
associated with permanent hearing loss when activated by exposure to excessive
noise or ototoxic agents; identify those environmental agents, drugs, etc. that
potentiate hearing loss from noise exposure and to characterize the degree and
extent to which the additional hearing loss occurs; study those specific non-
auditory systems (endocrine, immunologic, physiologic, pharmaca'ogic, teratogenic,
cardiovascular) that may be affected by chronic noise exposure; identify, by
appropriate epidemiological methods, those factors that are related to hearing
loss and the non-auditory effects of noise.

Areas under current investigation include: investigation of the correlation
between the cochlear potentials and ion movements within the cochlea and their
alteration by exposure to noise; examination of the quantitative differences
between damage caused by impact and steady state noise when equated for total

170



energy content; studies on the physiological mechanisms underlying the cochlear
damage caused by cis-dichlorodiammine platinum (II); identification of the physio-
logical mechanisms corresponding to complex signal analysis (speech) breakdown
after slight noise trauma not predictable from simple signal pure tone tests;
verification of technical factors affecting the precision of current speech
discrimination tests and the development of electronic hardware to correct some of
the sources affecting the precision; and noise effects upon the embryo and fetus.

The studies on the role of ionic permeability of the endolymph-perilymph barrier
in normal and noise exposed guinea pigs have continued. Exposure to noise caused
a decrease of K permeability in the cochlear partition which was clearly corre-
lated with the suppression of hair cell activity. Exposure to noise was found to
alter the K permeability of the organ of Corti selectively. These results were
further supported by our findings that intracochlear application of tetraethyl-
ammonium suppressed hair cell function. Using intracellular recording of hair
cells of the organ of Corti, it^was found that maintenance of the hair cell
function was dependent on the K conductance of the apical membranes ^hair cells^
The endolymph-perilymph barrier was found to be less permeable to Na than to K .
Exposure to noise did not result in substantial changes in Na permeability of the
endolymph-perilymph barrier. The movement of CI" in the cochlear fluids was
coupled with entry of K into the endolymph so as to maintain electroneutral ity of
the endolymph. The rate constant for water of the endolymph-perilymph barrier was
also examined in normal guinea pigs in conjunction with measurements of osmolarity
of the cochlear fluids. The preliminary results showed that the rate constant for
water was substantially greater than that for K or CI".

The effects of continuous and impact noise on cochlear potentials were compared in
guinea pigs. Our results indicated that the degree of cochlear response suppres-
sion produced by ;mpact and continuous noise of equal energy was not equivalent
under short-term exposure conditions. The degree of cochlear response suppression
produced by long-term exposure will be studied in guinea pigs with chronically
implanted round-window electrodes.

The ototoxicity of cis-dichlorodiammine platinum (II) was studied in guinea pigs.
Multiple administration of cis-DDP resulted in a marked suppression of the cochlear
microphonics but little change in the endocochlear potential was observed. The
suppression of hair cell activity was more pronounced in the lower turns of the
cochlea. There was a close corrleation between loss of hair cells and suppression
of cochlear microphonics. No marked changes in electrolyte concentrations in the
cochlear fluids were observed in cis-DDP treated guinea pigs.

An electronic auditory nerve simulator has been constructed for use in shakedown
and testing of various strategies for computerized for generating trinary pseudo
random noise was developed, since the binary noise conventionally used to test
auditory system transfer function was recently shown to generate 2nd order distor-
tion of the same type as the auditory system. A general purpose (computer based)
transfer function analysis program accepting analog inputs while outputting
transfer function magnitude and phase, cocherence, input and output power spectra,
auto and cross correlation and impulse or step response has been developed.
Extension of the transfer function program to accept nerve fiber histogram outputs
is being pursued.

Experiments to determine the effect of in utero noise exposure on the conceptus
and fetus have continued. The exposure of pregnant CF-1 mice to either semi-
continuous high level noise (126 dBA jet engine noise) or unanticipated high

171



intensity startling sounds, resulted in embryo-lethal ity and decreased pregnancy
maintenance. This effect did not appear to be related to elevation of plasma
coritcosterone levels. No teratogenic effects were noted in CF-1 mice in response
to noise exposure. Exposure of the CF-1 mouse to noises, whose spectra were
coincident with the most acute frequency band of the mouse (18-20 KHz), resulted
in significant late stage fetolethality, an effect previously shown by others to
be correlated with exogenous catecholamine application. Replication of the
experiment in CD-I mice with concomitant measurement of catecholamine levels in
plasma and uteri failed to generate late stage fetolethality. Teratogenic effects,
lowered pregnancy rate, excess early stage resorption, and lowered maternal and
fetal weight were noted. Plasma norepinepherine/epinepherine ratio was reversed
by noise exposure in late stage pregnancy and uterine norepinepherine levels were
significantly elevated during late stage pregnancy in noise exposed CD-I mice.
This experiment also revealed a previously unreported universe relationship
between uterine NE content and plasma corticosterone and progesterone concentration
during the postimplantation period. In another experiment exposure of pregnant
guinea pigs to textile mill noise (elevated to 115 dB SPL) caused a significant
deterioration ring of the offspring as measured by the brainstem evoked response
techniques.

In conjunction with the Laboratory of Behavioral and Neurological Toxicity, the
effects of maternal noise exposure on neurobehavioral parameters during adult life
were assessed. Maternal exposure resulted in neuromotor (grip strength, swim
endurance) differences in both male and female offspring as adults. An effort to
identify the mechanisms involved in this result is underway.

A version of a fiber optic lever optimized for impact measurments of mechanical
surfaces has been developed. This version, which should be very useful in diagnosing
noise emission from complex machinery, is being used by others as the vibration
sensing component of an acoustic intensity meter. Fiber optic motion sensor
technology intended for use in investigating basilar membrane signal conversion
characteristics using a twist-etch bottle coupler terminated in a single 50 y
fiber is being pursued. Techniques for fiber etching, sputtering gold film on the
45° fiber tip, and acquiring sufficient bandwidth from a one nanowatt optical
return signal have been developed.

As part of an investigation into factors affecting precision of current speech-
perception-in-noise tests, an instrument was developed which measures all relevant
speech electro-acoustic amplitude parameters. Since provision for generating and
modulating white, pink, and speech spectrum noise was also included, the instru-
ment can be utilized to investigate interaction of the masking noise spectra
characteristics and intelligibility; including the potential for confounding test
scores. Since the major technical capabilities have been assembled in a single
instrument for the first time, this development is expected to stimulate further
investigation, perhaps leading to progress in assessing the social and economic
cost of hearing loss. Progress in quantifying hearing loss relative to the
difficulties experienced in everyday noisy environments is presently impeded by
the imprecision of conventional methodology and technology. The instrument has
been used in this laboratory in assessing intelligibility of speech passed through
hearing aids and novel speech transduction devices having less distortion. In
these tests, scores obtained with speech mechanically coupled onto the ossicular
chain and electronically recovered from the cochlea by differential electrode
techniques significantly exceeded those obtained with similar speech passed
through hearing aids. Additional tests showed that, where distortion of the peak
clipping type was involved, the conventional speech peak equalization methodology

172



'acted to confound test scores while use of the long term RMS amplitude measure
did not.

The technology and experience gained in these tests are being further exploited
through recording of test tapes designed to quantify the potential for noise
spectra and intermodulation distortion confounding. Data acquisition from normal
and hearing damaged groups is to be obtained by others colaborating with this
laboratory.

LIGHT

This program is concerned both with the biological effects of artificial lighting
and with the interactions that occur between light and chemical agents in the
skin (photosensitization). The beneficial effect of sunlight in the photoactiva-
tion of vitamin D precursors in the skin is well known. Cyclical changes in
lighting also affect the maturation of gonads in both mammals and man. In addition
to these effects sunlight elicits a number of undesirable side effects ranging
from erythema ("sunburn") to skin cancer. More recently it has been suggested
that artifical light sources, particularly those which have energy spectra that
are markedly different from sunlight, may have undesirable side effects. For this
reason, we have carried out a series of studies to determine whether fluorescent
lights and high pressure sodium vapor (HPSV) lamps have any hitherto unknown
biological effects. Sprague-Dawley rats were born and reared under either
daylight-simulating fluorescent lights or HPSV lamps. Animals housed under the
HPSV lamps had heavier adrenals, smaller gonads (males only), larger kidneys
(females only) and elevated red and white cell counts. No differences were
observed between the two groups in the swim endurance, tail flick and hotplate
tests.

Another adverse effect of light results from its interaction with chemical
agents in the skin. The chemical agent may be endogenous (e.g., protoporphyrin),
a drug (e.g., sulfonamides, declomycin), topical agent (e.g., p-aminobenzoic acid
in sunscreens) or an environmental agent (e.g., polycyclic aromatic hydrocarbons).
The combined effect of light and these agents causes skin photosensitization
which may take the form of either phototoxicity or photoallergy. While the
initial step in all forms of photosensitivity must be the absorption of light by
the chemical or its metabolites, the precise mechanism is unknown. Irradiation
of several sulfonamide drugs in aqueous and organic solution has been shown to
produce a variety of carbon, sulfur and nitrogen-centered free radicals. Data
suggest that the sulfur trioxide anion radical (S03*~) may be involved in the
photosensitivity induced by these compounds. The phototoxicity of the anti-
inflammatory drug benoxaprofen appears to result from the generation of oxygen-
centered reactive species including superoxide, singlet oxygen and peroxy radicals.
Irradiation of methanol ic solutions of musk ambrette (2,6-dinitro-3-methoxy-4-t-
butylbenzene) generated two nitro anion radicals that further decompose to other
photoproducts.

The photodegradation of polycyclic aromatic hydrocarbons absorbed onto solid
phases is the subject of a grant. In this work, the photochemistry of particulate
adsorbed polycyclic aromatic hydrocarbons is being examined in a fluidized bed
reactor irradiated by a xenon arc lamp. In this system the half life of pyrene
adsorbed to glass particles is about 160 minutes. Six different pyrene photo-
products have been isolated using high pressure liquid chromatography.



173



MOLECULAR BIOPHYSICS

The Molecular Biophysics Program is conducting research to understand at the
molecular level the interaction of environmental agents with target biological
systems including nucleic acids, proteins membranes and microsomal systems. For
these studies a number of highly sophisticated spectroscopic techniques, e.g.,
electron spin resonance and nuclear magnetic spectroscopy, fluorescence and
absorption spectroscopy, circular dichroism and stopped flow spectrometry, are
being employed.

Nucleic Acids:

It is now widely recognized that many mutagenic agents exert their biological
effects by modifying DNA. The covalent binding of chemically reduced adriamycin
and daunorubicin to DNA has been examined. Results show that, under identical
conditions, one adriamycin molecule is bound per 15 nucleotides whereas only one
daunorubicin is bound per 140 nucleotides. These findings may explain why
adriamycin induces more DNA damage than daunorubicin as evidenced by an increase
in sister chromatid exchange. Enzymatical ly activated drugs also bind covalently
to DNA with identical binding ratios. Results with synthetic polynucleotides
show that the binding takes place predominately at guanine bases of DNA. In vivo
binding studies show that adriamycin binds to DNA, RNA and proteins, however,
binding decreases rapidly with time suggesting an enzymatic repair process is
operative. The active alkylating agents derived from these drugs are not known
at this time. Other investigations have also shown that several antitumor drugs
induce membrane protein conformational changes in erythrocyte ghosts and mastocytoma
cells. This finding suggests that the cytotoxic and mutagenic properties of
these agents may involve membrane effects as well as interaction with nucleic
acids. Recently, Moore has proposed a mechanism for the bioactivation of adriamycin
to a covalent binding species. In this scheme, a quinone methide with a carbo-
cation character at Cy is presumed to be alkylating species. We had earlier
suggested that, in addition to the quinone methide, a Cy free radical inter-
mediate may also act as an alkylating agent. We have recently examined the
formation and binding of these species from adriamycin and daunorubicin to DNA.
Our studies indicate that the microsomal -NADPH activation produces both one
electron (Cy-free radical) and two electron reduction (Cy-quincne methide)



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1982) → online text (page 14 of 90)