The Toxicologist - Society of Toxicology

5.00 and 7.50 mg/kg/day by oral gavage; the high dose group was sacrificed on Day
15 due to excessive morbidity/mortality). Animals also were evaluated for micronucleated
RETs (mnRETs) by FCM, DNA damage using the comet assay (blood,
liver, stomach), and chromosome aberrations (CAbs) in peripheral blood lymphocytes
(PBLs). All endpoints were analyzed at two or more timepoints where possible.
Statistically significant (p< 0.05), dose-dependent increases were observed for:
Pig-a mutant RBCs at 7.50 mg/kg/day on Day 15 and 5.00 mg/kg/day on Day 29;
Pig-a mutant RETs at 5.00 mg/kg/day on Day 15 and 2.50 mg/kg/day on Day
29; and mnRETs at 2.50 mg/kg/day on Day 29. Responses were consistently
higher at the later timepoints, indicating accumulation of genetic damage with repeat
dosing. The largest increases observed (fold-increase compared to control,
where significant; all at 5.00 mg/kg/day on Day 29) were: mutant RETs
(21X)>mutant RBCs (9.0X)>mnRETs (2.0X). In contrast, no such increases were
observed for CAbs in PBLs (Days 4 and 29), DNA damage in liver (Days 15 and
29) or stomach (Day 29), or DNA damage in PBLs (Days 1, 15 and 29). Since
4NQO is a well known mutagen, clastogen and carcinogen, the lack of response for
some of the endpoints was unexpected. A follow-up study is in progress to evaluate
the same endpoints using single acute doses equal to the 28-day cumulative totals,
or using the same total doses split over three days as in a typical Comet/micronucleus
combination assay. The acute treatments were well tolerated, and half of the
treated animals will be followed for up to 28 days after treatment to evaluate persistence
of any observed responses.
332 CASE STUDY ON DOSE-RESPONSES FOR
MUTAGENICITY AND CLASTOGENICITY INDUCED
BY DNA-REACTIVE CHEMICALS.
L. H. Pottenger 1 , M. M. Moore 2 , T. Zhou 3 and E. Zeiger 4 . 1 The Dow Chemical
Company, Midland, MI, 2 U.S. FDA/NCTR, Jefferson, AR, 3 U.S. FDA/CVM,
Rockville, MD and 4 Errol Zeiger Consulting, Chapel Hill, NC.
Genotoxicity is a key regulatory driver in selecting the dose-response model used
for a cancer risk assessment, especially for chemicals acting by a mutagenic mode of
action (MOA). Debate continues over the utility of data to inform the shape of
low-dose dose-response curves. In 2009, NAS recommended a linear dose-response
model as the most likely best fit to describe both cancer and non-cancer data due to
human population variability. In recent years, published examples of directly DNAreactive
chemicals with non-linear dose-response curves for genotoxicity have increased.
A multi-stakeholder effort was initiated to evaluate published data for
genotoxic dose-response characterization using in vitro and in vivo studies of several
directly DNA-reactive chemicals: ethylene oxide, ethyl- and methyl methanesulfonate,
ethyl- and methylnitrosourea, and acrylamide(/glycidamide). This review,
based on a mutation MOA (Pottenger & Gollapudi, 2010), focused on biomarkers
of exposure (DNA adducts) and effect (mutations; micronuclei). Such surrogate reporter
effects (e.g., Hprt, micronuclei), designated as biomarkers of early effect and
not on a path leading to cancer, are nevertheless useful to inform the dose-response
of a chemical with a mutagenic MOA. The review concludes that, while the
strength of the evidence varies among the chemicals, in part due to methodological
issues or inadequate dose coverage, as a whole there is a solid database, one which
includes statistical determination of best fit dose-response models in some cases,
supporting the conclusion that directly DNA-reactive chemicals can demonstrate
non-linear/threshold dose-response relationships for induction of mutation and/or
clastogenicity. This analysis provides significant support to the conclusion that, for
some carcinogens where the induction of mutation is an early key event (directly
DNA-reactive, mutagenic MOA), the overall dose-response for a mutagenic MOA
for cancer can also be non-linear. (Does not represent FDA policy.)
333 HIGH-THROUGHPUT IN VITRO MICRONUCLEUS
ASSAY: AUTOMATED SCORING OF 384-WELL PLATES.
S. Bryce, S. Avlasevich and S. Dertinger. Litron Laboratories, Rochester, NY.
This laboratory has described a flow cytometric method for scoring in vitro micronuclei
(MN) [Environ. Molec. Mutagen. (2006) 47:56-66]. This In Vitro
MicroFlow® method labels necrotic and mid-/late-stage apoptotic cells with ethidium
monoazide. Cells are then washed, stripped of their cytoplasmic membranes,
and incubated with RNase plus a pan-nucleic acid dye. Here, we report modifications
to the method whereby all procedures are accomplished in the same 384-well
plate. For these experiments, CHO-K1 or human TK6 cells were treated continuously
for 24 - 30 hrs with a diverse range of genotoxic and non-genotoxic chemicals
(51 chemicals tested in CHO, 29 in TK6). For these experiments, each plate included
7 compounds at 20 concentrations in duplicate. With the assistance of a
high throughput sampler flow cytometric analyses occurred automatically (2,500
cell equivalents per well). Since a consistent number of latex particles were added to
each specimen, nuclei to bead ratios were collected concurrently with MN frequencies,
and these data were used to derive relative survival measurements. Dose-dependent
MN induction was observed for 27 of the 28 genotoxic agents studied in
CHO, and 22 of the 23 genotoxicants studied in TK6. Two instances of false-positive
results were observed with the non-genotoxicants. These data were collected at
a rate of up to 5 plates, or 1920 wells, per day. Collectively, these data suggest that
in vitro MN screening can be accomplished in a miniaturized and highly automated
format. Further work is needed to characterize assay sensitivity and specificity with
a greater number of chemicals, and also to assess the transferability of the method
through inter-laboratory trials.
334 PHOTO-CLASTOGENICITY TESTING IN HUMAN
PERIPHERAL BLOOD LYMPHOCYTES.
S. Roy, H. Murli, M. Chan, L. F. Stankowski Jr. and T. Lawlor. Genetic &
Molecular Toxicology, Covance Laboratories Inc., Vienna, VA. Sponsor: M. McKeon.
UV light is known to activate certain classes of chemicals to forms that can be clastogenic
to living cells. In order to detect such chemicals, photo-clastogenicity assays
are routinely performed using various transformed cell lines. However, transformed
cell lines are genetically unstable by nature and can affect the sensitivity and specificity
of the assay since many of their cellular functions are compromised as compared
to normal primary cells. Therefore, we have evaluated the use of primary
human peripheral blood lymphocytes (HPBLs) from healthy donors for this purpose.
Venous blood was collected and isolated lymphocytes were PHA-stimulated
to grow in mass culture for 48 hours. Individual 10 mL cultures were prepared and
treated in 100-mm dishes with 8-methoxypsoralen (8-MOP; 0.10-0.60 μg/mL), or
the saline and mitomycin C (0.50 μg/mL) controls. One set of cultures was exposed
to a Xenon arc solar simulator lamp with a UV filter (290 nm cut off), while
the other set was kept in dark. UV doses ranged from 106 mJ/cm2 - 211.9 mJ/cm2
for UVA and 12.3 mJ/cm2 - 24.7 mJ/cm2 for UVB. All cultures were then incubated
in the dark for 3 hours, after which treatments were terminated and cells were
grown in fresh culture medium for another 20 hours. Cells were harvested and
slides prepared, and chromosome aberrations were analyzed as described in OECD
testing guideline 473. 8-MOP induced statistically significant (2 to 3-fold) increases
in percent aberrant cells in the presence of photoactivation, but no increases
were observed in the absence of solar simulated light. These results indicate that
photo-clastogenic chemicals can be detected in primary HPBLs. Further validation
is ongoing with additional photo-genotoxic chemicals.
335 PCB3 METABOLITES INDUCE SPINDLE
ABERRATIONS AND REDUCE TUBULIN
POLYMERIZATION.
S. Flor and G. Ludewig. University of Iowa, Iowa City, IA.
Recently, semivolatile lower chlorinated biphenyls (PCBs) have been identified as
significant air pollutants in inner city air, in indoor air of schools and other buildings
from the 1950s - 1970s, and at other sites. Inhalation exposure to these compounds
is of great concern in light of new studies revealing that at least one such compound,
4-monochlorobiphenyl (PCB3), has tumor initiating and mutagenic activity in rats.
PCB3 is readily metabolized to mono- and dihydroxy-biphenyls (PCB3-Cat, PCB3-
HQ) and further to quinones (PCB3-oQ, PCB3-pQ). We reported that in cells in
culture PCB3-HQ induces polyploidization and all 4 metabolites produce micronuclei,
with exception of the PCB3-pQ predominantly through chromosome loss. To
gain further insight into the mechanism of this damage induction we analyzed mitotic
phase distribution and spindle morphology in V79 Chinese hamster lung cells
and tubulin polymerisation in vitro. All 4 metabolites induced a significant increase
in monoaster formation of the spindle, in the case of PCB3-HQ with outstanding
efficacy, i.e. nearly ¾ of the mitotic cells showed monoaster formation at 7.5 μM.
PCB3-pQ had a high potency, ¼ of mitosis showed monoasters at 3 μM. PCB3-Cat
induced a wide range of spindle disturbances at the highest concentrations tested
(10 and 15 μM), including an increase in tripolar meta- and ana-phase mitosis and
multipolar spindles, which was also observed in cells treated with its redox-partner
PCB3-oQ (10 μM). The tubulin polymerization assay revealed that PCB3-HQ did
not interact with tubulin at concentrations up to 500 μM, whereas all other compounds
reduced tubulin polymerization by destabilization of the microtubules. Our
results suggest that the formation of monoasters could be involved in the extensive
tetraploidization observed with PCB3-HQ, whereas aberrant spindle formation and
inhibition of tubulin polymerization may play a role in chromosome loss-induction
by these PCB3 metabolites. (Supported by NIEHS P42 ES013661 and DOD
DAMD17-02-1-0241)
SOT 2011 ANNUAL MEETING 71