The Toxicologist - Society of Toxicology

1702 DOES THE CLOCK MAKE THE POISON? INFLUENCE
OF THE CIRCADIAN CLOCK ON TOXICOLOGICAL
MECHANISMS AND OUTCOMES.
H. Zarbl 1 and L. A. Hooven 2 . 1 Environmental and Occupational Health Sciences
Institiute, Robert Wood Johnson Medical School, Piscataway, NJ and 2 Department of
Zoology, Oregon State University, Portland, OR.
The daily biochemical, physiological, and behavioral activities of many organisms
are synchronized by light/dark cycles. These temporal oscillations are maintained
by the circadian clock, which has intrinsic periodicity of approximately 24 hours.
The circadian rhythm of cells is controlled by the interaction of multiple positive
and negative feedback loops comprising a molecular oscillator, which modulates expression
through transcriptional and epigenetic means. Environmental and occupational
exposures leading to disruption of circadian rhythm, including jet lag, lightat-night,
and shift work are associated with an increased risk of endometriosis,
breast cancer, and prostate cancer, prompting the International Agency for Cancer
Research to classify shift work as a probable human carcinogen (type 2A).
Disruption of the circadian clock is also associated with increased risk of cancer,
cardiovascular disease, diabetes, obesity, reproductive problems, sleep disorders,
drug and alcohol addiction, and psychiatric disorders. Accumulating evidence indicates
that the dynamic influence of the circadian clock profoundly affects many
critical pathways in toxicology. Exposure to stressors, carcinogens, chemotherapeutic
agents, and other xenobiotics can alter circadian function in cells, rodents, and
humans, while some chemopreventive agents my reset the rhythm. Targeted disruption
of clock gene expression is advancing our ability to understand and manipulate
the circadian clock. Our panel of experts will review the state of this emerging
area and explore opportunities for disease prevention.
1703 ENVIRONMENTAL INFLUENCES UNCOUPLE
PERIPHERAL CLOCKS FROM SCN CLOCKS.
U. Schibler. Molecular Biology, University of Geneva, Geneva, Switzerland. Sponsor:
H. Zarbl.
Cell-autonomous and self-sustained circadian oscillators are operative in virtually
all body cells of mammalian organisms. These remarkably robust clocks must be
synchronized periodically by a master circadian pacemaker residing in the suprachiasmatic
nucleus (SCN) of the brain to keep phase coherence. This is accomplished
through a variety of signaling pathways employing blood-borne factors (e.g. cyclically
secreted hormones and growth factors), metabolites, body temperature, and
neuronal cues. I shall present experimental strategies for the identification of signaling
pathways that participate in the synchronization by feeding cycles, oscillating
blood-borne signals, and body temperature rhythms. NAD+-sensing enzymes, such
as Sirtuin deacetylases and poly(ADP-ribose)polymerase 1 (PARP-1) are likely candidates
for conveying the metabolic state of a cell to its circadian oscillator. We
demonstrated that both SirT1 and PARP-1 indeed impart on circadian gene expression,
and that PARP-1 participates in the feeding-dependent synchronization
of liver circadian clocks. A novel technology, dubbed STAR-PROM (for Synthetic
Tandem Repeat Promoter, to screen for all immediate early transcription factors
(IETFs) that serve as sensors of blood-borne signals revealed 14 artificial promoters
containing binding sites for IETFs that respond differentially to human and/or rat
plasma samples collected at three- to four-hour time intervals around the clock.
These IETFs are candidates for being components of signaling pathways that are
stimulated by cyclic systemic blood-borne signals regulated directly or indirectly by
the circadian master pacemaker in the SCN. Simulated mouse temperature
rhythms with an amplitude of only 8,5% (min. 35 °C, max. 38 °C) are also capable
of synchronizing the clocks of cultured fibroblasts. Using MEFs from the relevant
knockout mice and by performing RNA interference in NIH3T3 cells we demonstrated
that both HSF1 and the cold-inducible RNA binding protein CIRP are required
for the efficient phase entrainment by body temperature rhythms.
1704 TICK-TOX: CLOCK GENE EXPRESSION AND
INTERACTIONS BETWEEN THE MOLECULAR
PATHWAYS FOR THE REGULATION OF CIRCADIAN
RHYTHMS AND TOXIN METABOLISM.
D. J. Earnest. Neuroscience and Experimental Therapeutics, Texas A&M Health
Sciences Center, College Station, TX. Sponsor: H. Zarbl.
Members of the Per-Arnt-Sim (PAS) family of transcriptional regulators are involved
in development and in sensing and adapting to environmental changes.
Most PAS proteins function as heterodimers consisting of a sensor protein complexed
with a general binding partner and through these interactions mediate biological
responses to environmental conditions. In this regard, the PAS protein, aryl
hydrocarbon receptor (AhR) partners with AhR nuclear translocator (ARNT) to
mediate the metabolism of drugs and environmental toxins. Because AhR and
ARNT are commonly expressed in the cells and tissues throughout the body with
other PAS genes in molecular feedback loops forming the mammalian circadian
clockworks, it is possible that functional interactions between these PAS proteinregulated
pathways may have implications for the biological consequences of xenobiotic
exposure. To examine the role of key PAS proteins in the circadian clockworks
in modulating xenobiotic metabolism, we determined whether the
toxin-mediated activation of the AhR signaling pathway in the mammary gland and
liver: 1) fluctuates rhythmically; and 2) is altered following targeted disruption or
siRNA inhibition of key PAS genes in the circadian clockworks, Period 1 (Per1) and
Per2. In both the mouse mammary gland and liver, the inductive effects of the prototypical
Ahr ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on a key target
of AhR signaling, Cyp1A1, were marked by diurnal variation such that TCDD-induced
Cyp1A1 expression was 23-43 fold greater during the night than during the
daytime. In mutant mice, targeted disruption of Per1 alone or Per1 and Per2
(Per1ldc and Per1ldc/Per2ldc) abolished this diurnal rhythm in the toxin-mediated
induction of hepatic and mammary gland Cyp1A1 expression and significantly increased
the TCDD-induced expression of p450 genes. These studies indicate that
the clock genes, Per1 and Per2, may function as inhibitory factors in the diurnal
modulation of AhR-regulated responses to toxins and thus have important implications
for intercommunication between these PAS protein-regulated pathways.
1705 THE HEPATOCYTE AUTONOMOUS CLOCK
MODULATES THE CHRONOTOXICITY OF
ACETAMINOPHEN.
C. A. Bradfield 1 , J. A. Walisser 1 , B. P. Johnson 1 , Y. Liu 1 , A. Shen 1 , E. L.
McDearmon 1 , B. McIntosh 1 , A. Vollrath 1 , A. C. Schook 2 and J. S. Takahashi 2 .
1 The McArdle Laboratory for Cancer Research, University of Wisconsin School of
Medicine and Public Health, Madison, WI and 2 Northwestern Univeristy, Evanston, IL.
The hepatotoxicity of Acetaminophen (APAP) was first reported in the 1960s and
its circadian changes in metabolism in the 1970s. The dose independent circadian
variation in APAP hepatotoxicity is thought to be primarily due to oscillations of
liver GSH and possibly changes in cytochrome p450 enzymatic activity which follow
eating patterns. The circadian clock drives the 24 hour oscillations in these factors
directly and indirectly (e.g. by direct regulation of drug metabolizing enzymes
or by driving feeding rhythms). Because the circadian clocks of various tissues can
become uncoupled or disrupted under conditions such as shift work, we set out to
determine the relative contributions of the central clock in the suprachiasmatic nucleus
(SCN) and the hepatocyte circadian clock in modulating the chronotoxicity
of APAP. Using mice with conditional null alleles of the Mop3 locus, we were able
to generate livers harboring hepatocytes with little or no cell autonomous circadian
rhythms. The observation that these mice become resistant to the chronotoxicity of
APAP suggests that while the central clock modulates detoxification indirectly
through feeding rhythms and GSH; the hepatocyte clock controls daily oscillations
in major aspects of APAP bioactivation by the cytochrome P450 system.
1706 CIRCADIAN EXPRESSION OF DRUG PROCESSING
GENES IN MICE.
C. D. Klaassen. Pharmacology, University of Kansas Medical Center, Kansas City, KS.
Temporal coordination of hepatic drug-processing gene (DPG) expression facilitates
absorption, biotransformation, and excretion of exogenous and endogenous
compounds. To further elucidate the circadian rhythm of hepatic DPG expression,
mice were subjected to a standard 12-h light/dark cycle, and livers were collected six
times a day. The mRNAs of hepatic phase I enzymes (cytochromes P450, aldehyde
dehydrogenases, and carboxylesterases), phase II enzymes (glucuronosyltransferases,
sulfotransferases, and glutathione-S-transferases), uptake and efflux transporters,
and transcription factors were quantified. In general, the mRNA of phase I
enzymes increased during the dark phase, whereas the mRNAs of most phase II enzymes
and transporters reached maximal levels during the light phase. The majority
of hepatic transcription factors exhibited expression peaks either before or after the
onset of the dark phase. During the same time period, the negative clock regulator
gene Rev-Erbα and the hepatic clock-controlled gene Dbp also reached mRNA expression
peaks. Considering their important role in xenobiotic metabolism, hepatic
transcription factors, such as constitutive androstane receptor, pregnane X receptor,
aryl hydrocarbon receptor, and peroxisomal proliferator activated receptor α, may
be involved in coupling the hepatic circadian clock to environmental cues. Taken
together, these data demonstrate that the circadian expression of the DPG battery
and transcription factors contribute to the temporal detoxification cycle in the liver.
SOT 2011 ANNUAL MEETING 367