Colorimetric Mutagenicity
Assay for Lead Optimization in Drug Development
Michael R. Schiedler, Yali Chen, Adams Amantana,
Shanthakumar TR, Dennis E. Hruby
Siga Technologies Inc.
The bacterial reverse mutation or Ames assay
is part of the standard ICH gentoxicity test
battery that evaluates the mutagenic potential
of a compound by assessing the ability of the
compound to induce reverse mutations (His- to
His+) in bacterial strains. Traditionally, the
assay is performed on minimal glucose agar plates
with and without cytochrome P450-mediated metabolic
activation of the test compound. To expedite
genotoxicity screening in drug development,
we have adapted a colorimetric mutagenicity
assay in a 96-well format. With modification
to "Environmental Bio-Detection Products
Inc" method, we developed an in-house testing
system that consists of four Salmonella tester
strains TA-98, TA-100, TA-1535, and TA-1537
with the corresponding positive mutagenic controls
(2-nitrofluorene , Sodium Azide , 9-amnioacridine)
and 2- aminoanthracene as a promutagen. The
tester strains are grown overnight in LB broth
at 37°C to an OD of 1.0. Each test is set-up
with a mixture of a test compound diluted in
DMSO and a sodium phosphate buffer containing
Davis-Mingioli salts, NADP, L-histidine, D-biotin,
D-Glucose, Glucose-6-phosphate and bromocresol
purple (pH indicator) and plated on a sterile,
non-tissue culture treated 96-well flat bottom
plate. In order to test the potential mutagenicity
of a metabolite of a compound, rat-liver S9
extracts are added. After incubating for 5 days
at 37°C, the plates are analyzed based on
a color change in culture media indicating a
shift in pH due to the accumulation of CO2 produced
by the growth of revertants of the tester strain.
The current protocol for the colorimetric mutagenicity
assay can accommodate screening for up to four
compounds of interest, at four dose levels,
in four Salmonella tester strains with and without
rat liver S9 in a single experiment. The system
has been proven to be a viable alternative to
the conventional Ames assay with comparable
sensitivity, specificity, and has reduced compound
quantity requirements. So far, more than a hundred
compounds have been prescreened with this in-house
system, and the results have facilitated rapid
screening for structure-genotoxicity relationship
studies. Its implementation has significantly
decreased the number of compounds requiring
the standard Ames assay and thus enhanced lead
optimization in our preclinical drug development.
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A high-throughput approach
for identifying disrupters of vitamin A signaling
Yanling Chen and David H. Reese
U.S. Food and Drug Administration
Retinoic acid (RA) is an important signaling
molecule in vertebrates. As an activating ligand
for transcription factors, RA controls, directly
or indirectly, the expression of more than 500
genes and is essential for normal embryonic
development and cellular function in adult animals.
RA is generated in cells of higher animals from
vitamin A (retinol; ROH) by two enzymatic reactions
in which ROH is oxidized to retinaldehyde (RAL)
and RAL to RA. Further oxidation converts RA
to polar metabolites that are eliminated to
maintain RA concentrations at cell-defined levels.
The pathway from ROH to RA and the regulation
of gene expression by RA, referred to here as
the vitamin A signaling pathway (VASP), is one
of the most important signaling pathways in
vertebrates and its disruption has adverse consequences
for the embryo and adult cells and tissues.
Many agents that interfere with the pathway,
causing either abnormally low or high cellular
levels of RA, are teratogenic. In adult animals,
vitamin A deficiency causes a range of adverse
effects including cognitive impairment, loss
of normal cell differentiation, and impaired
reproduction, vision, and immune function.
We are developing an in vitro, high-throughput
assay for the rapid detection of chemicals that
interfere with the expression of genes that
are regulated through the VASP and thus have
the potential to be fetal and adult toxicants.
The assay uses a mouse pluripotent EC stem cell
line (P19) that has a completely functional
VASP. It can be induced to differentiate into
nerve cells by RA, a capacity it shares with
cells of the developing nervous system. Nerve
cell differentiation, in vitro and in vivo,
is dependent on the upregulation of homeobox
gene expression. A 40-50 fold increase in the
expression of one of these genes, Hoxa1, can
be detected two hours after the addition of
ROH. This assay uses rapid RT-QPCR to detect
chemicals that interfere with the ability of
ROH to regulate the expression of Hoxa1. It
can detect chemicals that interfere with the
production of RA from ROH; it also detects chemicals
that interfere with the metabolism RA.
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Incorporating flow cytometry
based automated scoring into the reconstructed
skin micronucleus assay
Erica L Dahl1, Greg Mun1, Steve Bryce2, Stephen
Dertinger2, Rodger Curren1
1Institute for In Vitro Sciences
2Litron Laboratories
The Reconstructed Skin Micronucleus Assay (RSMA)
using EpiDerm tissues (MatTek Corporation) has
been developed as a possible replacement for
in vivo genotoxicity testing of cosmetics, which
is now banned in Europe by the Seventh Amendment
to the Cosmetics Directive. The assay is currently
undergoing evaluation for interlaboratory reproducibility
in the United States and Europe as part of an
international validation effort. The assay performs
well for correctly identifying positive and
negative genotoxins, but scoring micronucleus
induction microscopically is extremely labor
intensive, which may limit widespread use of
the assay. We have investigated the feasibility
scoring micronuclei from the RSMA by flow cytometry
to increase the speed at which the data are
acquired as well as to improve the precision
of the assay. EpiDerm tissues were treated in
triplicate with 0, 1, 3, 10 or 30 micrograms
per milliliter of Mitomycin C in acetone for
48 hours using our standard protocol. Following
treatment, single cell suspensions were prepared
from the basal cell layers of the EpiDerm tissue
constructs, and cells were processed for flow
cytometry using the Litron In Vitro MicroFlow®
kit. Single cell suspensions from tissues treated
in parallel with cytochalasin B and the same
concentrations of mitomycin C were affixed to
slides and stained with Acradine orange for
microscopic analysis. The results from In Vitro
MicroFlow® analysis were comparable to those
from standard microscopic scoring, demonstrating
that this automated scoring platform can be
used to quantify genetic damage in a 3-dimensional
epidermal tissue construct. Since flow cytometry
is already widely used by many laboratories
and its efficiency and objectivity are well
known, this development has the potential to
increase the usability of the RSMA.
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Assessment of micronucleus
induction by amorphous silica nanoparticles
in vivo and in vitro
Kyle P. Glover1, Christie M. Sayes2, Abby
Myhre1, Kenneth L. Reed1, Keith A. Swain3, Michele
Ostraat4, E. Maria Donner1, David B. Warheit1
1DuPont Haskell Global Centers, Newark, DE
2Texas A&M University, College Station,
TX
3DuPont Company, Wilmington, DE
4Research Triangle Institute, Research Triangle
Park, NC
The potential risk associated with the quickly
advancing production of nanomaterials has created
an intense debate among toxicologists. Some
in vitro assays including the comet and Cytochalasin
B micronucleus assay have established evidence
of genotoxicity. However, studies analyzing
the in vivo genotoxic effects of nanoparticles
are scarce. The purpose of our in vivo experiment
was to assess the induction of micronucleated
reticulocytes in rats exposed to aerosolized
amorphous silica nanoparticles. The assay was
an additional endpoint on another study in the
spirit of the 3 Rs (Refinement, Reduction, and
Replacement) of animal use, and the data generated
are used for in vitro comparisions. Male Crl:CD(SD)rats
were exposed for 1 or 3 days, 6 hours per day,
to aerosolized amorphous silica nanoparticles
(37 nm or 83 nm) at concentrations ranging from
3.1 x 107 to 1.8 x 108 particles/cm3. Control
animals were exposed to air. Peripheral blood
samples were collected 24 hours post exposure.
Additional samples were collected at 1 week
and 1 month post exposure. The collected blood
samples were fixed in ultra cold methanol (-80oC)
and analyzed by flow cytometry according to
the In Vivo MicroFlow Plus Rat Micronucleus
assay kit (Litron Laboratories, Rochester, NY).
Approximately 20,000 reticulocytes were analyzed
per animal with 2-5 animals per testing condition.
For both the 30 nm and 72 nm particles sizes
at 1 and 3 day exposures there was no statistically
significant increase in micronucleated reticulocytes
over the negative control animals. These data
were supplemented with an in vitro assessment
of non-aerosolized amorphous silica using the
In Vitro MicroFlow Micronucleus assay kit (Litron
Laboratories, Rochester, NY) in CHOK1 cells.
In vitro cytotoxicity endpoints (LDH and MTT)
as well as inflammatory cytokine release (TNF-?
and IL-6) were also analyzed in rat alveolar
epithelial cell line L2 and the rat alveolar
macrophage cell line N8383.
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In vivo Rat Pig-a Mutation
Assessment by Flow Cytometry: Inter-Laboratory
Comparison of ENU Treated Rats
William C. Gunther1, Ronald D. Fiedler1,
Dean Franklin2, Souk Phonethepswath2, Raja
Sarojini2, Thomas J. Shutsky1, Catherine J.
Thiffeault1, Stephen D. Dertinger2
1Pfizer Global Research and Development
2Litron Laboratories, Inc
This laboratory has been evaluating the performance
of a recently developed in-vivo Pig-a gene mutation
assay in rat peripheral blood following treatment
with genotoxic agents. Briefly, Pig-a and other
genes are required in the formation of Glycosylphosphatidylinostidol
(GPI) anchors that bind proteins to the cell
surface of erythroid cells. In wild-type cells
these surface proteins can be labeled with fluorescent
dyes and measured by flow cytometry. A mutation
at the pig-a locus can result in dysfunction
of the GPI anchor and thus an absence of cell
surface protein and signal. These mutant cells
are enumerated with the wild-type cells to determine
the mutant frequency. In addition, since the
assay differentiates reticulocytes from mature
erythrocytes the mutant frequency can be determined
in each specific sub-population of erythrocytes.
In this study individual experiments performed
in ENU treated rats at Pfizer and Litron Laboratories
were compared to evaluate the portability of
the assay. Results herein show a remarkable
consistency of data and overlapping dose-response
trend across multiple sample times was generated
between the two labs which indicates the assay
is readily transferable to across independent
laboratories.
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Measurement of Multiple Genotoxic
In Vivo Endpoints using the Comet, Micronucleus
and Pig A mutation Assays
William C. Gunther, Stephanie L. Coffing,
Donna A. Dickinson, Maria E. Engel, Ronald D.
Fiedler, Susan D. O'Lone, Kelley E. Sanok, Maik
J. Schuler, Thomas J. Shutsky, Catherine J.
Thiffeault, Bethany A. Traverso
Pfizer Global Research and Development
The current revision of the ICH guideline (S2R)
emphasizes the use of in vivo genotoxicity testing
for the purpose of primary hazard identification
and as a follow-up to in vitro positive findings.
In order to reduce animal testing, integration
of Genetic Toxicology endpoints into subchronic
toxicology studies would be preferred, which
raises the question as to which endpoints and
tissues should be evaluated. Traditional in
vivo Genetic Toxicology hazard identification
of DNA damage includes acute endpoints like
the in vivo micronucleus and the in vivo comet
assay. By comparison, the recently developed
Pig-A mutation assay evaluates the genotoxic
potential in blood erythrocytes and allows for
accumulation of DNA damage over time. The objective
of this study is to compare and contrast the
dose-response and time course for the induction
of acute (micronucleus, comet) and chronic accumulative
(Pig-A) DNA damage in the blood cells from the
same rats following subchronic 28-day treatment
with the genotoxic agents ethyl nitrosourea
(ENU) and ethyl methanesulfonate (EMS). In addition,
after terminal harvest, the comet and in vivo
micronucleus assay will be used to assess DNA
damage in the liver and the GI tract. The results
will be discussed in the context of the integration
of genotoxicity endpoints into standard toxicology
studies.
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Different Approaches To
Automation Of The In Vitro Micronucleus Assay
Using Image Analysis
Michael L. Homiski, Elizabeth Rubitski, Andrew
Scott, Jennifer Munzner
Pfizer Global Research and Development
The in vitro micronucleus assay is well established
as an early screen to evaluate the genotoxic
potential of drug candidates by some pharmaceutical
sponsors and is currently under consideration
by regulatory agencies as a suitable alternative
to the in vitro chromosome aberration (CA) assay
and mouse lymphoma assay. At Pfizer we have
over twelve years of experience using microscopic
evaluation of the cytokinesis-block in vitro
micronucleus assay (CBMN) to screen drug candidates
and have recently reported 86.6% concordance
with the CA assay. In addition we have over
four years of experience using an automated
in vitro micronucleus platform, developed in
house to measure micronuclei induction in mononucleated
cells. This prescreen assay is used at very
early stages of drug development and is highly
sensitive (>99% of prescreen positive compounds
are CBMN positive) but due to the inherent limitations
of the platform, specificity is lower with a
potential of up to 30% false negatives. Since
commercially available automated CBMN platforms
were insensitive for detecting weak pharmaceutical
positives, we developed a second system capable
of examining micronuclei in binucleated cells.
This automated platform is state of the art
with a QC interface that is fast, reliable and
used to confirm the classification of micronucleated
cells and we fully anticipate it to out perform
the manual microscopic CBMN assay. Based on
our experience, the advantages and disadvantages
of these in vitro MN screens will be compared
and contrasted within the context of supporting
the early development of drug candidates.
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Use of Cultured Human Skin
Cells and A Skin Tissue Construct as Models
to Study the Genotoxic Marker Y-H2AX Following
Sulfur Mustard Exposure
Adele L. Miller, C.L. Gross, E.W. Nealley,
O.E. Clark, N.K. Waraich, K.L. Rodgers, W. J.
Smith
USAMRICD
Sulfur mustard (2-2'-dichlorodiethyl sulfide,
SM) is a cytotoxic chemical warfare agent. The
skin serves as a principal target site for in
vivo toxicity of SM exposure resulting in the
formation of blisters and inflammation. To elucidate
genotoxic effects of SM, Normal Human Epidermal
Keratinocytes (NHEK, Lonza Corp., MD) and a
commercially available, multicellular skin tissue
construct, EpiDerm™ (MatTek Corp., MA),
served as in vitro models to observe the presence
of Y-H2AX foci. Y-H2AX is a phosphorylated derivative
of the H2AX histone and is tightly bound to
double stranded DNA break sites. In its phosphorylated
state, H2AX can be used as a reproducible indicator
of genotoxic injury. Cells and constructs were
exposed to 0, 50, 100 and 300 ?M concentrations
of SM for 2 and 24 hrs. Following exposure,
tissues underwent fluorescent immunohistochemistry
using mouse anti-?-H2AX antibody. Propidium
iodide (PI) was used as a nuclear stain. NHEKs
were fixed, permeabilized and incubated with
mouse anti-?-H2AX antibody. Cells were analyzed
for ?-H2AX via flow cytometry. Our results show
that SM exposure results in the formation of
?-H2AX foci and this change is prevalent at
300 ?M SM. This indicator of DNA damage will
be a useful biomarker for the study of SM toxicity
and therapy.
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Refining Mutagenicity Structural
Alerts
Glenn J. Myatt, Dave Bower, Kevin Cross,
Donald Quigley
Leadscope
There are multiple public sources describing
genetox structural alerts. These alerts have
been consolidated and used to analyze a database
of high quality genetox information containing
over 9,500 compounds and 150,000 test results
using the Leadscope software. The database includes
endpoints for the different Salmonella strains.
Statistics have been generated to help understand
how well these alerts are able to classify genetox
compounds. For structural alert classes where
there is sufficient data, such as aromatic nitro
compounds, a systematic analysis was performed
to understand what structural features modulate
the activity.
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Successful transfer of
cytotoxicity assessment by flow cytometry (FCM)
confirms superior reproducibility of mitotic
index (MI) measurements
Daniel J. Roberts1, Spellman, RA2, Sanok,
K2, Chen, H1, Chan, M1, Yurt, P1, Thakur, AK1,
Devito, GL2, Murli, H1, Stankowski Jr., LF1
1Covance Laboratories, 9200 Leesburg Pike, Vienna,
VA 22182
2Pfizer Global R&D, Eastern Point Road,
Groton, CT 06340
Prevention of non-relevant positive results
for the in vitro chromosome aberration assay
depends, at least in part, upon the accuracy
of cytotoxicity measurements. A FCM procedure
for determining MI, developed and utilized routinely
at Pfizer, has been adopted successfully by
Covance. This method, using antibodies against
phosphorylated H3 (S10) in the presence of nucleic
acid cross-staining, has been evaluated at the
two independent test sites and compared to manual
scoring. Primary human lymphocytes were treated
with cyclophosphamide, mitomycin C, benzo(a)pyrene
and etoposide at concentrations inducing dose-dependent
cytotoxicity. Deming's regression analysis indicates
that the results generated via FCM were more
consistent between sites than those generated
manually. Further analysis using the Bland-Altman
modification of the Tukey mean difference method
supports this finding, as the standard deviations
of differences in MI generated by FCM were less
than half of those generated manually. Decreases
in scoring variability due to the objective
nature of FCM and the greater number of cells
analyzed makes FCM a superior method for MI
measurements. Additionally, the FCM platform
has proven to be transferable and easily integrated
into standard genetic toxicology laboratory
operations.
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Evaluation of 26 Chemicals
using two Automated In Vitro Micronucleus Image
Analysis Platforms against the Proposed OECD
Guideline 487
Elizabeth Rubitski, Maria Engel, Michael
Homiski, Maik Schuler, Jocelyn Sherman, Karrie
Tartaro, Claudia Wiersch
Pfizer Global Research and Development
The In Vitro Micronucleus (IVMN) assay has been
used for years as a screening tool to predict
the aneugenic and clastogenic potential of chemical
agents. Regulatory agencies are currently considering
the IVMN assay (Proposed OECD Guideline 487)
as an alternative for both the GLP (Good Laboratory
Practices) In Vitro Cytogenetics and Mouse Lymphoma
assays. Since there are advantages to running
the assay both with and without cytochalasin-B
(CYB), e.g. to increase sensitivity and specificity
for certain classes of compounds, and we have
an in-house image analysis system with two IVMN
platforms, capable of evaluating micronuclei
in both mononcleated (-CYB) and binucleated
(+CYB) Chinese Hamster Ovary cells, we plan
to evaluate extensive dose-response relationships
for 26 commercially available chemicals. Thirteen
chemicals included in our study are suggested
in the guideline for validation and all 26 will
be evaluated in 3 independent tests. Here we
report the preliminary range finding cytotoxicity
results within the context of each IVMN platform
(+/- CYB) and the OECD Proposed Guideline 487.
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Acute and Sublethal Effects
of Lindane (Edc) in a Common Carp Cyprinus Carpio
with Special Reference to Haematological and
Biochemical Studies
Manoharan Saravanan, Kuppusamy Prabhu Kumar,
Mathan Ramesh
Unit of Toxicology, Department of Zoology, Bharathiar
University, India
The acute and sublethal effects of an endocrine
disruptor compound (EDC) lindane, a synthetic
organochlorine pesticide on haematological parameters
like red blood corpuscle (RBC), white blood
cell (WBC), haematocrit (Hct), haemoglobin (Hb),
mean cellular volume (MCV), mean cellular haemoglobin
(MCH), mean cellular haemoglobin concentration
(MCHC); biochemical parameters such as plasma
glucose, plasma protein and glycogen levels
in liver and muscle of a freshwater fish Cyprinus
carpio were studied in this present study. The
median lethal concentration of lindane for 24
h was 0.38 ppm. During acute (24 h) treatment
hemoglobin, hematocrit and erythrocyte values
were decreased in the lindane exposed fish registering
a percent decrease of 82.05, 78.48, and 54.93,
respectively, whereas the leucocyte value increased
(85.52 %). The other hematological indices like
MCV, MCH and MCHC were decreased. The biochemical
profiles like plasma glucose and protein levels
were increased in lindane exposed fish showing
a percent increase of 50.36 and 3.98, however
the glycogen level in liver (21.70 %) and muscle
(1.74 %) were decreased when compared to that
of the control group. In the sublethal (0.038
ppm) treatment, hemoglobin and hematocrit values
were decreased up to 10th day and after that
increased throughout the treatment period of
35 days. A significant decrease in RBC count
and increase in WBC count was observed throughout
the study period. MCV value was decreased up
to 10th day after that it was increased throughout
the study period. The MCH (except 5th day) and
MCHC (except 15 and 25th day) values were increased
in lindane treated fish throughout the study
period. Plasma glucose level was significantly
increased and plasma protein level showed a
declining trend throughout the study period.
Glycogen level in liver and muscle of fish Cyprinus
carpio exposed to lindane for 35 days were decreased
up to 10th day and then glycogen level was increased;
at the same time the muscle glycogen decreased
up to 15th day and it was increased up to the
end of experimental period. The present study
showed that the pesticide as well as endocrine
disruptor compound lindane caused alterations
in certain haematological and biochemical parameters
of C. carpio at the acute and sublethal concentration
levels.
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Development of a High Throughput
in vitro Micronucleus Assay using High Content
Imaging Technology
Sampada A. Shahane, Menghang Xia
NIH Chemical Genomics Center, National Institutes
of Health, 9800 Medical Center Drive, MSC 3370,
Bethesda, Maryland, USA
Micronucleus assay is a short term mammalian
cell mutagenic assay, which has been widely
used for assessment of genotoxicity of chemicals.
This assay can detect compounds causing genetic
damage resulting in the formation of small membrane
bound DNA fragments called Micronuclei in the
cell cytoplasm. Existing gold standard in vitro
micronucleus assay uses manual counting and
thus is time consuming, requires several trained
people and can be subject to lot of variance.
Thousands of cells per treatment need to be
scored by more than one person to have greater
statistical significance. This makes the evaluation
more tedious and time consuming. We used Image
Xpress Micro, a High Content Screening instruments
from MDS analytical technologies that has proprietary
micronucleus module for automatic evaluation.
Using CHO K1 cells in 96-well format we evaluated
assay with Mitomycin C and Cyclophosphamide
in absence or presence of S9 (metabolic activation)
conditions. A total of 14 concentrations in
addition to the basal were tested in duplicates
of which top 7 were scored manually and all
were scored automatically. Unlike manual scoring,
automated scoring rapidly and consistently generates
a lot of data using low amounts of compounds.
This feature can make automated micronucleus
scoring assay a suitable candidate for screening
large number of potential environmental genotoxic
compounds.
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The Influence of Metabolic
GSTM1, GSTT1 AND GSTP1 on DNA damage in pesticide
exposed workers
Satyender Singh1, Vivek Kumar2, Sachin Thakur1,
Basu Dev Banerjee2, Shyam Sunder Grover1, Devendra
Singh Rawat1, Syed Tazeen Pasha3, Sudhir Kumar
Jain1, Shiv Lal1, Arvind Rai1
1National Institute of Communicable Diseases,
India
2University College of Medical Sciences &
GTB Hospital
3National Programme for Prevention and Control
of Fluorosis
Pesticides pose a clearly identifiable risk
to those who are occupationally exposed to this
carcinogen causing acute and chronic health
impacts including various neoplastic diseases
and congenital malformations. Studies have indicated
that pesticides are metabolized by various xenobiotic
metabolizing enzymes including glutathione S-transferases
(GSTs). GSTs provide critical defense against
carcinogens and their polymorphism has been
found to be associated with various forms of
cancer. Genetic polymorphism that affects xenobiotic
metabolism or cellular response to DNA damage
can modulate individual sensitivity to genotoxins.
Therefore, it is important to identify the potential
genetic susceptibility factors affecting individual
responses to carcinogen exposure. Hence, the
present study was designed to evaluate the genotype
frequency of GSTM1, GSTT1, GSTP1 and their influence
on DNA damage in pesticide exposed occupational
workers. Using the Comet assay, the extent of
DNA damage was evaluated in the PBMC of 70 pesticides
exposed workers and equal number of age and
gender matched control subjects. The metabolic
genotype in GSTM1/GSTT1 (null deletion) and
GSTP1 (Ile105Val) were identified using PCR-RFLP.
The results showed that pesticide exposed subjects
had significantly greater comet parameters than
the control subjects as measured through olive
tail moment (p<0.001, 95% CI= 5.48-6.56),
DNA tail length (p<0.0001, 95% CI= 22.10-25.51),
tail extent moment (p<0.0001, 95% CI= 18.51-20.80)
and % tail DNA (p<0.0001, 95% CI= 15.79-18.56),
respectively. The prevalence of GSTM1 (p=0.57,
OR= 0.75, ?2= 0.31), GSTT1 (p=0.83, OR= 1.19,
?2= 0.04) and Ile105Val GSTP1 (p=0.80, OR= 1.26,
?2= 0.05) did not differ significantly in pesticide
exposed subjects compared to controls. Further
analysis showed significant difference in the
DNA comet parameters carrying GSTP1 Ile-Ile
as compared to those carrying both GSTP1 Ile-Val/Val-Val
(p=0.009, 95% CI= 0.84-5.67). In conclusion,
the results suggest that occupational workers
with GSTP1 Ile-Ile homozygote may be at increased
risk of DNA damage but not with null deletion
of GSTM1, GSTT1 and GSTP1 Ile-Val/Val-Val due
to pesticide exposure.
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Genetic Toxicity Assessment
of the Nitroxide Radical 2,2,6,6 Tetramethylpiperidin-l-oxyl
(TEMPO)
Graeme B.J. Smith1, Ramadevi Gudi2, Valentine
O. Wagner III2, Wannie Madraymootoo2, Frans
Van Velsen3, Jacky Van Gompel4, Anita Bigger5
1Vertex Pharmaceuticals Incorporated
2BioReliance Corporation, Department of Genetic
Toxicology
3Tibotec BVBA, Preclinical Development
4Johnson & Johnson, Global PreClinical Development
5FDA-CDER, Division of Antiviral Products
The commonly employed process reagent and potential
process impurity 2,2,6,6-tetramethylpiperidin-l-oxyl
free radical (trade name TEMPO) was evaluated
for genotoxic potential following a review of
the available and somewhat conflicting published
data. Evidence of a positive mutagenic response
in an exploratory screening and a definitive
GLP-compliant bacterial reverse mutation assay
was limited to equivocal findings of a 2.1-fold
maximum increase in revertants noted only in
the GLP-compliant study with Salmonella typhimurium
tester strain TA100 in the presence of S9 at
5000 µg/plate which was not confirmed
in a follow-on repeat assay. Evidence of a positive
clastogenic response noted in a GLP-compliant
chromosome aberration assay using Chinese hamster
ovary (CHO) cells involved a statistically significant
increase (19.0%) in percentage of cells with
structural aberrations noted only at 250 µg/mL
[highest dose scored based on toxicity: 54%
cell growth inhibition relative to solvent control
as determined by relative increase in cell count
(RICC) with a 30.6% reduction in relative cell
count (RCC)] in the initial non-S9-activated
4 hour exposure group. In a follow-on confirmatory
assay under identical exposure conditions, 225
µg/mL (highest dose scored based on toxicity:
54% cell growth inhibition determined by RICC
with a 28.5% reduction in RCC) resulted in a
non-statistically significant increase (5.5%)
in percentage of cells with structural aberrations
which fell just outside the historical solvent
control range (0.0 - 5.0%). In an effort to
conduct a thorough investigation, the 250 µg/mL
dose level was also scored (even though the
cell growth inhibition reached 73% determined
by RICC with a 39.3% reduction in RCC) and resulted
in a statistically significant increase (15.0%)
in percentage of cells with structural aberrations.
Results of these evaluations suggest that the
genotoxic potential of TEMPO may be associated
with a threshold-based effect. At concentrations
above a defined threshold, observed DNA damage
may be occurring as a result of cytotoxicity,
saturation of anti-oxidant defense mechanisms,
and/or TEMPO's potential pro-oxidant effects
noted previously at higher (millimolar) concentrations.
Additional experiments are planned to investigate
the potential threshold-based effects described
above.
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OECD/GLP Compliant In Vitro
Micronucleus Assay in TK-6 Cells
Zhanna Sobol, Jennifer R. Cheung, Stephanie
L. Coffing, Paula A. Muehlbauer, Maik Schuler,
Andrew Scott, Richard A. Spellman
Pfizer Global Research and Development
The Organization for Economic Co-operation and
Development (OECD) has proposed a guideline
for harmonizing the in vitro micronucleus assay.
This study explores the performance of human
lymphoblast TK-6 cells in the cytokinesis-blocked
in vitro micronucleus assay (CBMN) in compliance
with draft OECD TG487 guidelines.
TK-6 cells are p53 proficient and have a better
regulated cell cycle and DNA repair mechanisms
than most rodent cell lines. As a result, the
use of this cell line may reduce resources required
to do follow-up testing on false positives.
An additional advantage is that TK-6 is a suspension
cell line and thus amenable to flow cytometry
analysis. A flow cytometric method has been
developed and utilized to assess toxicity parameters
and determine dose selection.
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Evaluation of Proposed
Cytotoxicity Endpoints for the In Vitro Mammalian
Micronucleus Assay in the Presence and Absence
of Cytochalasin B in CHO and TK6 Cells
Catherine Thiffeault, Robert Boyes, Jennifer
Cheung, Donna Dickinson, Maria Engel, Jocelyn
Sherman, Maik Schuler
Pfizer Global Research and Development
The in vitro micronucleus assay has been extensively
used as an in vitro screening tool for test
articles that might have aneugenic or clastogenic
potential. Currently, the Organisation for Economic
Co-operation and Development (OECD) is working
toward a final version of a guideline for the
conduct of the in vitro mammalian micronucleus
assay but a few questions regarding appropriate
cytotoxicity measurements and cytotoxicity limits
remain. In order to resolve the remaining questions,
we compared the induction of micronuclei at
the top dose (50 - 60% cytotoxicity) determined
by either Relative Cell Counts (RCC), Relative
Increase in Cell counts (RIC), Relative Population
Doublings (RPD), or Cytokinesis-Blocked Proliferating
Index (CBPI) using three weak and strong inducers
of micronuclei in both the presence and absence
of cytochalasin B (CYB) in Chinese hamster ovary
and human lymphoblastoid TK-6 cells. In order
to assess extensive dose-response relationships,
we selected a number of expected weak (diazepam,
quinacrine hydrochloride, phenolphthalein) and
strong (vinblastine sulphate, mitomycin C, cytosine
arabinoside) inducers of micronuclei with a
variety of different mechanisms of action for
testing. The results clearly demonstrated that
all six compounds produced positive responses
using either cytotoxicity measurement. Surprisingly,
the weak inducers of micronuclei generally produced
higher responses in the assay without CYB than
under the same treatment conditions with CYB.
The outcome from these studies further supports
the cytotoxicity measurements and cytotoxicity
limits proposed in the draft OECD guideline.
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The Use of Dimethoxyethane
as the Vehicle for Ames Assays
Valentine O. Wagner, Renee M. Hines, Emily
W. Dakoulas, Melissa R. VanDyke, Anissa S. Wiley,
Brian M. Taylor
BioReliance Corporation
Data published in the late 1990's supported
the use of dimethoxyethane (DME) as a vehicle
in the Ames assay; however, these studies did
not use the full complement of tester strains
required by OECD. Testing DME in the battery
of strains as recommended by the guidelines
will allow use of this vehicle in a GLP Ames
assay. Several exploratory studies were conducted
in our laboratory, using the plate incorporation
method and tester strains TA98, TA100, TA1535,
TA1537 and WP2 uvrA. Each strain was tested
both with (10%) and without S9 activation using
25, 50 and 100 µL aliquots of DME. In
addition, the standard positive controls were
exposed in both the presence and absence of
DME. No toxicity was exhibited with DME at 25
to 100 µL per plate with any of the tester
strains in either the presence or absence of
S9 activation. The vehicle control values were
also consistent with the historical vehicle
control values. In addition, the positive control
values in the presence of DME were consistent
with the concurrent positive control values
in the absence of DME as well as the historical
positive control values. Based on these results,
DME can be used as the vehicle in the plate
incorporation method at 25 to 100 µL per
plate.
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Screening for Potential
Genotoxic Metabolites using Electro-optical
and Nanoreactor-LC/MS Arrays
Linlin Zhao1, James F. Rusling1,2, John
B. Schenkman2
1Department of Chemistry, University of Connecticut
2Department of Cell Biology, University of Connecticut
Health Center
Genotoxic metabolites represent a potential
stumbling block in drug and environmental chemical
development. Thus, it is quite important to
assess potential reactive metabolites of drug
candidates in order to predict toxicities early
in the discovery pathway. We have developed
a two-tier molecular-structure based genotoxicity
screening approach involving in-vitro electro-optical
arrays and nanoparticle bioreactors that generate
damaged DNA for LC-MS analysis. Both the arrays
and the nanoparticles feature thin films of
oxidative and conjugation enzymes, DNA, a metallopolymer
that produces increased light emission when
reacting with damaged DNA. Herein, we demonstrate
proof-of-concept for these approaches in assessing
potential genotoxic metabolites via various
single and multi-enzyme activation and detoxication
pathways. Specific examples include (1) cytochrome
P450 mediated activation and UDP-glucuronyltransferase
detoxication of tamoxifen, (2) N-acetyltransferase
mediated activation of 2-aminofluorene, and
(3) glutathione S-transferase mediated activation
of ethylene dibromide. The first tier screen
involves the arrays, in which elevated light
intensity observed when molecules are bioactivated
suggests DNA adduct formation. In the second
tier, DNA/enzyme nanoparticles are used to elucidate
structural details and formation rates of specific
DNA adducts by LC-MS in 96-well plate format.
The array and LC-MS thin film approaches serve
as a molecular-structure based general platform
to screen potential genotoxic metabolites, and
as such are complementary to batteries of existing
toxicity bioassays.
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Mutagenicity of Organic
Extracts of Raw Water, Pre-chlorinated Water,
and Finished Water from the Huangpu River
Weiwei Zheng1, Li Chen2, Ying Zhou1,
Xia Wang1, Songhui Jiang1, Weidong Qu1
1Department of Environmental Health, School
of Public Health, Fudan University, Shanghai,
China
2Center for Disease Control & Prevention
of Hongkou, Shanghai, China
Organic compounds in row water that have not
been eliminated could form new DNA-damaging
agents after chlorination. The Huangpu River
is a major freshwater source of drinking water
for millions of Shanghai residents. Because
domestic sewage and industrial wastewater are
continually poured into this river, the quality
of drinking water prepared from the raw water
of this river is of concern to the public and
governmental regulatory agencies. The present
study was aimed to evaluate the mutagenecity
of organic extracts of raw, pre-chlorinated,
and post-chlorinated water from the Huangpu
River with Ames test and to identify potential
mutagens in post-chlorinated water with gas
chromatography/mass (GC/MS). Organic compounds
in 50 L of raw (untreated), pre-chlorinated,
or post-chlorinated (finished) water were absorbed
by macro-reticular resin columns (Amberlite
XAD-2) and then desorbed with 30% acetone in
methanol by 15 ml per min. The extracts were
further dissolved in DMSO for testing. The mutagenic
activity of the organic extracts was tested
using Salmonella typhimurium strains TA98 and
TA100 with or without S9 mix, at doses of 0.5,
1.0, and 2.0 L water equivalent/plate. In addition,
organic compounds in the finished water were
analyzed by GC/MS. The Ames test showed that
the mean number of his+ revertant colonies in
all extract-treated groups was more than 3 times
greater than that in the negative control group
in TA98 with or without S9. There was a dose-dependent
increase in the number of revertant colonies
treated with extracts from raw, pre-chlorinated,
or post-chlorinated water and the number of
colonies was highest in the post-chlorinated
water groups. No significant increases in the
frequency of revertant colonies caused by any
extracts were observed in TA100 with or without
S9. Compared to previous results using the same
test in 1980s, it appeared that the mutagenicity
of raw or finished water tested in this study
was stronger. Among the 142 kinds of organic
compounds in 10 categories detected in the finished
water using the EPA 525 methods, N-phenyl-?-naphthylamine
(a potential carcinogen considered by NOISH),
dibutyl phthalate (DBP), and 4-cumylphenol were
found at relative high levels. Both DBP and
another identified compound, dioctyl phthalate
(DOP), were in the priority list of hazardous
substances to control recommended by EPA. Herbicides,
including atrazine and linuron, and estrogen
(3-deoxyestradiol) were also detected in the
finished water. The results indicated that organic
extracts of raw, pre-chlorinated, and post-chlorinated
water prepared from the Huangpu River were mutagenic,
with or without metabolic activation, and chlorination
enhanced the mutagenicity of organic compounds
in water. The GC/MS results can further provide
clues to identify the potential mutagens in
drinking water. Alternative water treatment
such as using active carbon to remove organic
compounds will provide high quality of drinking
water for the public.
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Effect of Glucose Concentration
In Minimal Glucose Agar (MGA) Medium On Growth
of Salmonella typhimurium TA97a in the mini-Ames
assay in 6-Well Plates
E. Sullivan, B. Martin, M. Mack, B. Mahadevan,
N. Collins, R. Snyder
Schering-Plough Research Institute
Periodically during the conduct of the mini-Ames
assay in 6-well plates, TA97a showed poor growth
under conditions which did not affect the other
tester strains. Testing of culture variables
including top agar source, and different incubation
times had no effect on bacterial growth. Since
TA97a is reported to be more highly sensitive
to glucose concentration than other Salmonella
strains, we studied the effects of MGA source,
composition, volume and age of plates. Poor
growth of TA97a was seen in agar plates approaching,
but not exceeding their given shelf lives. To
test the relationship between increased glucose
concentration and diminished colony counts,
both TA97a and TA100 were cultured on MGA plates
containing between 0% and 50% glucose (the traditional
amount is 20%). Bacteria were also cultured
in the presence and absence of an S9 metabolic
activation system. The TA100 cultures revealed
normal colony counts in all of the glucose concentrations
tested as compared to the historical control
values. For the TA97a cultures grown in the
presence of S9, there were diminished colony
counts at 30% glucose and no growth was detected
at higher concentrations. The TA97a cultures
grown in the absence of S9 showed no growth
above 25% glucose. In addition, normal colony
appearance, optimal size and number was detected
at 5-10% glucose. Despite the presence of a
background lawn, there were no colonies observed
with 0% glucose. Based on these findings it
was concluded that increased glucose concentration
in the MGA medium dramatically effects the growth
of TA97a in the mini-Ames assay using the 6-well
format.
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