The Toxicologist - Society of Toxicology

permitting treatment of cells on membranes (dry-wet cultures) with aerosols. We
used human cell models of different complexity: the lung tumor cell line H322,
normal human bronchial epithelial cells (NHBEC) and mini organ cultures of
nasal mucosa (MOC). As a model aerosol we applied smoke of 2R4F research cigarettes
generated according to ISO3308:2000. Even distribution of the smoke inside
the chambers was demonstrated by resazurin-assay. Carbon monoxide was monitored
on line in the atmosphere of the exposure units. Cultures were treated with
undiluted fresh smoke of 5 cigarettes for 50 min and were kept in an incubator afterwards.
CO concentrations exceeded 300 ppm after 12 min and persisted until
the end of the exposure period. In experiments with H322 cells viability was measured
by MTT-assay and additionally glutathione (GSH)-content was determined
by HPLC. All experiments included controls that were exposed to filtered air only.
Right after exposure viability was reduced to 93% and it declined to 91, 80 and
57% at 24, 48 and 72h post exposure. GSH-content was increased 1.4 fold after
24h and 1.9 and 2.1 fold at 48 and 72h post exposure. NHBEC were more sensitive
in cytotoxicity tests, as exposure to the smoke of 3 cigarettes for 33 min already
reduced viability to 93%. We also determined the GSH-content of MOCs exposed
to cigarette smoke. In contrast to the results in H322 cells GSH-content in MOCs
was reduced to 39 and 25% 24 and 72h after exposure, respectively. These experiments
showed the applicability of the evaluated cell culture exposure system to
study the effects of aerosols on cell models of different complexity.
984 COMPARATIVE REPRODUCTIVE AND
DEVELOPMENTAL TOXICITY OF AFLATOXIN B1 IN
THE WILD TYPE AND MUTANTS OF NEMATODE
CAENORHABDITIS ELEGANS.
L. Tang 1 , S. Xue 2 , G. Qian 1 , H. Ma 1 , P. L. Williams 1 and J. Wang 1 .
1 Environmental Health Science, University of Georgia, Athens, GA and 2 University of
Texas at Austin, Austin, TX.
Aflatoxin B1 (AFB1), a fungal metabolite in food, is one of the most significant
mycotoxins. Its potent hepatotoxic and carcinogenic effects involve metabolic activation
of parent compound through cytochrome P450 (CYP450) enzymes. The reproductive
and developmental toxic effects of AFB1, although were shown in certain
animal models, have not been well studied. The nematode C. elegans has
emerged as an excellent model for toxicological studies due to its short life cycle.
Because the complete genome sequence and cell lineage map of C. elegans have been
identified, genetic manipulation of genes, especially for CYP450 encoding genes,
can be made. In this study, toxic effects of AFB1 on the development and reproduction
of the wild type and mutant C. elegans were investigated. N2 (wild type),
VC40 (CYP13A7 gk31 del), VC603 (CYP14A2 gk289 del), and VC249
(CYP14A5 gk152 del), either at 1-day or 3-day old, were treated with AFB1 concentrations
at 0-16 mg/L under non-induced and β-naphthoflavone-induced conditions.
There are no significant differences on 24 h or 72 h lethality between wild
type and mutant C. elegans as evidenced by 24 h LC50 of 77.1, 55.9, >100, and
>100 mg/L AFB1 and 72 h LC50 of 4.7, 3.9, 5.1, 6.3 mg/L AFB1 in N2, VC40,
VC603, and VC249, respectively. AFB1 strongly disturbed the development and
reproduction of both wild and mutant C. elegans. The worm growth, expressed as
development arrested rate (DAR), which equals to (length of control - length of
treated)/length of control x 100%, was severely suppressed at AFB1 concentration
of 2 μg/mL and above. DAR was 28.4%, 17.1%, 12.4%, and 30.5% for N2,
VC40, VC603, and VC249 at 2 μg/mL AFB1, respectively. The reproduction was
also significantly affected by AFB1 with 24 h EC50 of 4.1, 1.4, 1.5, and 1.8 μg/mL
in N2, VC40, Vc603, and VC249, respectively. Analysis of AFB1 metabolites in
medium and worms by HPLC showed different chromatogram patterns between
treated and untreated with AFB1 among wild and mutant C. elegans.
985 β-CATENIN ACTIVATION IS NECESSARY FOR
SURVIVAL OF HUMAN BRONCHIAL EPITHELIAL
CELLS EXPOSED TO CIGARETTE SMOKE
CONDENSATE.
W. W. Polk. Research and Development, Lorillard Tobacco Company, Greensboro,
NC. Sponsor: C. Carter.
Bronchial epithelial cells reside in areas of the lung that receive high doses of exposure,
undergo significant changes in cell morphology, attachment and motility, and
may give rise to non-small cell lung carcinomas (NSCLC) following exposure to
cigarette smoke condensate (CSC). A screen of pharmacologic inhibitors in the
human bronchial cell line, BEAS-2B, revealed that inhibition of β-catenin decreases
the LD100 of CSC from 100μg/mL to 50μg/mL. Since β-catenin is a
known proto-oncogene and is a pathological marker strongly correlated with aggressive
tumor behavior in several cancers, we hypothesized that β-catenin activation
by CSC exposure increases the expression of genes associated with aggressive-
210 SOT 2011 ANNUAL MEETING
ness in NSCLC tumors. Using a multiplexed high content screening assay we
demonstrated that CSC treatment, in a dose and time dependent manner, increased
β-catenin activation. Furthermore, β-catenin activity was selected for, as all remaining
cells treated with 50μg/mL CSC or greater had activated β-catenin, whereas
control groups and low CSC doses had lower and variable activation of β-catenin.
A PCR array of CSC-treated cells demonstrated that the expression levels of genes
associated with aggressive NSCLC phenotypes through changes in extracellular matrix
remodeling, including the osteopontin, osteonectin and the MMPs are regulated
by β-catenin following CSC exposure. This work demonstrates that in Beas-
2B cells, CSC-induced β-catenin activation is an early survival response that
regulates the expression of multiple genes associated with increasing tumor aggressiveness
in models of NSCLC.
986 MOLECULAR EFFECTS OF INORGANIC AND
ORGANIC MERCURY IN CAENORHABDITIS ELEGANS
AND HUMAN CELLS.
M. K. McElwee and J. H. Freedman. NIEHS, Durham, NC.
Humans are exposed to both inorganic and organic mercury. While the toxicity of
mercury is well established, little is known about how different species act at the
molecular level. To address this issue, we employed a toxicogenomics approach
using the nematode C. elegans. Using sub-, low- and high-toxic exposures to inorganic
(HgCl2) and organic (CH3HgCl) mercury, the effects of these mercurials on
the steady-state mRNA levels for the entire genome were determined. A total of
473 and 2,865 genes were differentially expressed in the HgCl2 and CH3HgCl
(MeHg) treatments, respectively. Hierarchical clustering, principal components,
and pattern analyses indicated that the transcriptional responses of the mercurials
were unique. The biological function of the up-regulated genes was examined in C.
elegans using RNAi. The effect of RNAi and mercurial co-exposure on C. elegans
growth was tested for 599 genes. Knock-down of 18 of these genes affected the
growth of mercurial-exposed C. elegans. Of these 18 genes, 2 affected growth in response
to both mercurials. ABCG2, BACE1, BACE2, CHKA, CHKB, ELOVL3,
ELOVL6, GCLC and PARG are human homologs of the C. elegans genes that affected
growth following mercurial exposure. The effect of sub-, low-, and hightoxic
treatments of both mercurials on expression of these genes was examined in
SK-N-SH, HepG2 and HEK293 cells. Of the 162 cell-gene-mercury-toxicity combinations
tested, 36 resulted in a significant change in gene expression. Of these, 24
were differentially expressed by only one mercurial. The effect on viability of these
genes in mercurial exposure was also examined in the cell lines using RNAi. Eleven
significant gene-mercury interactions were found. There was not a cell type-gene
combination in which exposure to both mercurials affected viability. In whole organism
and cell culture studies, HgCl2 and MeHg showed unique effects on transcription
and different genes mediated the cellular response. These data indicate
that molecular mechanisms of toxicity differ by mercurial.
987 3D CELL CULTURE IMPROVES DETECTION OF
OXAZOLIDIONE TOXICITY.
J. Yang 1 , X. Xu 1 , K. Howe 2 , J. Dykens 2 and Z. Cui 1 . 1 Department of Engineering
Science, University of Oxford, Oxford, United Kingdom and 2 Drug Safety Research
and Development, Pfizer, Sandwich, United Kingdom. Sponsor: M. Sharpe.
Cell based assays are routinely used to detect potential cytotoxicity of novel compounds
during pre-clinical drug development. Conventionally such assays are performed
in static monolayer cultures. However, such 2D culture formats lack the 3D
structure of the real cellular environment and fail to accurately detect adverse effects
of many known human toxicants. To simulate in vivo reality we have been using
3D culture formats with media flow-through capabilities. Using a bioreactor developed
at the University of Oxford, we have grown rat adipose-derived stem cells
(ADSCs) in a 3D structure under a continuous flow of medium to maintain pH
and nutrition. Cytotoxicity of oxazolidiones via inhibition of mitochondrial protein
expression is induced by isomers with cis (CONA-04806), but not trans
(CONA-04807), stereo chemistry on the heterocycle. Using Alamar blue as a viability
index, we evaluated cytotoxicity of the two stereoisomers in 2D vs. 3D culture
formats with or without media flow. The isomer with cis stereochemistry was
significantly more cytotoxic than the trans isomer in both 3D-perfused or 3D-static
culture compared to either 2D-perfused or 2D-static culture. This effect was seen
after three days of treatment and continued to six days of treatment. The trans molecule
had no such effect with viability being similar in all conditions tested.
However, a reduction in cell viability was observed at the highest dose tested (30
μM) in both 2D and 3D culture. The results demonstrate that the 3D perfused system
improved the prediction of drug toxicity over 2D culture formats thereby recommending
3D culture for preclinical drug toxicity testing.