The Toxicologist - Society of Toxicology

1968 A HUMAN HEPG2 LUCIFERASE ASSAY FOR
METABOLICALLY ACTIVATED HEPATOTOXINS AND
GENOTOXINS.
X. Liu, K. A. Jeffrey, Y. Hu, J. M. Schmidt, J. Jiang and A. G. Wilson. Drug
Metabolism, Pharmacokinetics, and Toxicology, Lexicon Pharmaceuticals, The
Woodlands, TX.
Hepatic toxicity remains of major concern for drug failure; therefore a thorough examination
of chemically induced liver toxicity is essential for a robust safety evaluation.
In this manuscript, we describe a highthroughput GADD45β reporter assay
for assessing potential liver toxicity. Most importantly this assay utilizes a human
cell line and incorporates metabolic activation. Our high-throughput assay relies
upon two different reporter genes cotransfected into the HepG2 cells. The gene encoding
Renilla luciferase is fused to the CMV promoter and integrated into the
mammalian genome to provide a control for cell numbers. The firefly luciferase
gene is fused to the GADD45β promoter and used to report an increase in growth
arrest and DNA damage. A dual luciferase assay is performed by measuring the firefly
and Renilla luciferase activities in the same sample. Results are expressed as the
ratio of the two luciferase activities; increases over the base level (control) are interpreted
as induction of the GADD45β promoter and evidence of stress responses
due to the xenobiotic treatment. This mammalian dual luciferase reporter has been
characterized with and without metabolic activation using positive and negative
control agents. We evaluated GADD45β based induction of luminescence using 57
model compounds such as isoniazid, valproic acid, troglitazone, flutamide, MMS,
araC, cyclophosphamide, etc. These test compounds included hepatotoxins and
DNA damaging agents; many of which are well-known reagents employed as controls
in hepatotoxicity tests. Our results showed a high level of concordance with
known hepatotoxicants, some of which require metabolic activation and are poorly
detected by standard in vitro assays. The GADD45 gene is also strongly inducible
by DNA damage; thus this assay may provide a means to detect both hepatotoxins
and genotoxins. The GADD45 promoter fused dual luciferase assay represents a
valuable addition for the armamentarium for the early detection of hepatotoxic
compounds.
1969 TOXICOGENOMIC STUDY OF MICROCYSTIN-LR IN
WISTAR HAN RATS.
L. Fomby 1 ,C.Sabourin 1 , N. Machesky 1 , J. Price 1 , M. Kasoji 1 , M. Wendling 1 ,
D. Bornman 1 ,M.Hejtmancik 1 ,S.Auerbach 2 ,M.Hooth 2 , M. Vallant 2 and N. J.
Walker 2 . 1 Battelle, Columbus, OH and 2 NTP, NIEHS, Research Triangle Park, NC.
Exposure to microcystin produces hepatotoxicity and this study examined acute
toxicity and the toxicogenomic profile in the liver. Male rats were administered a
single intravenous dose of 0, 1, 10, 50, and 100 μg/kg microcystin –LR in sterile
PBS. Selected tissues (blood, liver, and kidney) were collected from 4 rats per timepoint
after 30 minutes and 1, 3, and 6 hours following dose administration.
Treatment with 50 and 100 μg/kg produced significant changes in the clinical and
anatomical pathology of the liver including elevated serum enzymes (ALP, ALT,
AST, and SDH), hepatocellular degeneration, and necrosis with congestion. Early
pathological changes were apparent at 30 minutes after treatment and necrosis
began to develop by three hours. Alterations in gene expression were apparent at
10, 50, and 100 μg/kg, with most changes occurring at 3 and 6 hours in the two
highest dosage groups. Up regulation of genes associated with liver damage and
necrosis occurred primarily in the 50 and 100 μg/kg groups. Activities of protein
phosphatase PP-1 and PP2A were decreased after the administration of at least 10
μg/kg. Differential expression of transcripts coding for PP1 and PP2A were up-regulated
in the 50 and 100 μg/kg groups at either 3 or 6 hours after exposure.
Microcystin exposure not only produces direct liver damage but also causes subtle
changes in toxicogenomic parameters associated with toxicity. This work was supported
by NIEHS Contract No. N01-ES-55536.
1970 INHALATION OF AMBIENT TRAFFIC RELATED
PARTICULATE MATTER DURING POSTNATAL LUNG
DEVELOPMENT INDUCES EARLY AND PERSISTENT
PULMONARY AND NEUROINFLAMMATION.
C. J. Johnston, L. Opanashuk, R. Gelein, J. N. Finkelstein, D. A. Cory-Slechta
and G. Oberdörster. University of Rochester, Rochester, NY.
Chronic airway disease and decreased lung function in children exposed to ambient
air pollution may be due to repeating cycles of injury and repair which alter normal
lung maturation. We hypothesized that children may be at higher risk to adverse effects
induced from air pollution as compared to adults because lung development is
a long term process and lung growth continues for an extensive postnatal period.
Children have a higher minute ventilation and activity and tend to spend more
time outdoors. In addition, increasing evidence suggests the inhaled pollutants can
similarly induce inflammation and oxidative stress in the brain. 4 and 56 day old
C57Bl/6J mice were exposed to ambient “Real World” ultrafine and fine particles.
Groups of 4 day old mice were exposed to ambient concentrated particles for 4
hours a day for 4 consecutive days and examined at the end of exposure or allowed
to recover until 8 weeks of age. Mice were either sacrificed or re-exposed to ambient
concentrated particles for 4 days and examined at the end of exposure. Mice exposed
starting at 4 days of age induced pulmonary mRNAs encoding p21 and
MnSOD. Re-exposure as adults induced mRNAs encoding proinflammatory cytokines
and chemokines. 4 day old mice exposed for two weeks, and recovered for 6
weeks exhibited evidence of brain inflammation, including the presence of reactive
microglia in the hippocampus and reactive astrocytes in ventral midbrain.
Inhalation of 10nm particles resulted in significant accumulation of gold nanoparticles
in Liver, Kidney, Spleen as well as three sections of the brain (Olf. Bulb,
Cerebellum and Cerebral Cortex) in both 4 and 56 day old mice. Developmental
exposure to ambient air pollution leads to sustained pulmonary and brain inflammation.
Our observations suggest that air pollutants may contribute not only to
respiratory disorders but may also predispose to persistent CNS dysfunction.
Funded By: EPA PM Center R-827354, U19 AI-067733, P30 ES-01247 and
NIEHS P01 ES11617
1971 CHRONIC LOW-LEVEL ARSENITE EXPOSURE AND ITS
EFFECTS ON CARDIOVASCULAR DEVELOPMENT
AND DISEASE.
P. Sanchez Soria 1 , D. Broka 1 and T. D. Camenisch 1, 2, 3 . 1 Pharmacology and
Toxicology, University of Arizona, Tucson, AZ, 2 BIO5 Institute, University of
Arizona, Tucson, AZ and 3 Steele Children’s Research Center, University of Arizona,
Tucson, AZ. Sponsor: J. Gandolfi.
Epidemiological studies have shown that a high incidence of cardiovascular diseases
and hypertension strongly correlate with elevated arsenic levels in drinking water.
An estimated 40% of developmental defects are caused by in-utero exposure to environmental
agents such as arsenic. Some of these defects can be fatal, but they are
also cause for altered gene expression, cell proliferation and differentiation, leading
to structural abnormalities and onset of diseases later in adulthood.
Our studies show that low level (50 and 100 ppb) arsenite in-utero exposure
through drinking water has an effect on proper heart development in our mouse
model, which could predispose the onset of cardiovascular diseases later in life. To
understand the mechanism of this alteration, in-vitro studies were done with real
time PCR on mouse primary heart organ cultures exposed to 10, 50, and 100 ppb
arsenite. Initial data show a decreased expression of genes related to epithelial-tomesenchymal
transition, a critical process in heart development. Arsenite treated
mouse embryos (E13.5) were sectioned and analyzed, showing abnormal development
of aortic and mitral valves, as well as ventricular myocardial thickening.
In in-vivo studies, blood pressure measurements are being obtained using a non-invasive
tail-cuff system. Age-matched FVB mice treated with 100 ppb arsenite are
then compared to controls over a period of one year. Preliminary results show an increase
in arsenite treated mice systolic (+31mmHg ± 12), and diastolic
(+23.7mmHg ± 10) blood pressures. Histological sections will be obtained after a
year-long treatment has been completed, and will be analyzed to determine potential
pathophysiological changes.
These results strongly suggest that arsenic exposure in-utero and early in life might
play a key role in the developmental basis and onset of diseases like hypertension
and other cardiovascular diseases. (NIH ES 04940; ES06694)
1972 GESTATIONAL LEAD EXPOSURE (GLE) PRODUCES
LATE-ONSET MALE-SELECTIVE OBESITY,
HYPERGLYCEMIA, AND PARA-INFLAMMATION: RISK
FACTORS FOR METABOLIC SYNDROME AND
NEURODEGENERATION.
D. A. Fox 1 , L. Leasure 1 , S. Chaney 1 , R. Hamilton 1 , R. Hao 1 , W. Xiao 1 , S.
Mukherjee 1 and J. E. Johnson 2 . 1 University of Houston, Houston, TX and
2
University of Houston-Downtown, Houston, TX.
Obesity is a pandemic as 65% of adolescents and adults are overweight and 30%
are obese. Epidemiological studies show a positive association between developmental
lead exposure and increased body mass index during adulthood. Recently
we reported that GLE produced dose-dependent, late-onset male-specific obesity.
Our goals were to measure food-fluid intake and characterize aging male and female
GLE offspring. C57BL/6 female mice were exposed to 55 ppm lead throughout
gestation and until postnatal day 10 (PN10): equivalent to human gestation period.
Male and female offspring were housed separately on PN21 and body weights
were taken at 1, 3, 6 and 9-14 months. Food-fluid consumption was measured
SOT 2010 ANNUAL MEETING 419