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Novel Interactions between Gut Microbiota
and Host Hepatic Xenobiotic
Biotransformation – Lessons Learned from
the Germ-free Mice
Julia Yue Cui, PhD
Assistant Professor, Sheldon Murphy Endowed Chair in
Toxicology and Environmental Health
Department of Environmental and Occupational Health
Sciences, University of Washington
Three examples of the role of gut microbiome in
regulating the hepatic drug-processing genes
• Gut microbiome and liver development
• Probiotics/conventionalization on hepatic drugprocessing gene expression
• Interactions between gut microbiome and environmental
chemicals
American College of Toxicology Webinar series
Slide 2
Liver P450 expression modulated by gut microbiota
Selwyn, Cheng, Bammler, Prasad, Vrana, Klaassen, and Cui JY*, 2015 Toxicol Sci, 147: 84-103
American College of Toxicology Webinar series
Slide 3
Protein expression was altered in livers of Germ-free Mice as
compared to Age-matched Conventional Mice
Selwyn, Cheng, Bammler, Prasad, Vrana, Klaassen, and Cui JY*, 2015 Toxicol Sci, 147: 84-103
American College of Toxicology Webinar series
Slide 4
Four Developmental Patterns of Critical Drug Processing Genes in
Livers of Conventional and GF mice
A
Selwyn, Cheng, Bammler, Prasad, Vrana, Klaassen, and Cui JY*, 2015 Toxicol Sci, 147: 84-103
American College of Toxicology Webinar series
Slide 5
Age and gut microbiota affect hepatic drugprocessing genes
• The absence of gut microbiota produced age-specific
effects on the regulation of hepatic drug-processing
genes
• The age-specific changes may be due to different
types/ratios of intestinal bacteria at various ages during
development in CV mice
American College of Toxicology Webinar series
Slide 6
Regulation of Hepatic Drug-metabolizing Enzymes in
Germ-free mice by Conventionalization and Probiotics
• Probiotics: live microorganisms that confer a health
benefit to the host when administered in adequate
amount
• VSL3 is a combinatorial probiotic that is used for human
intestinal disorders, such as inflammatory bowel disease
and ulcerative colitis
American College of Toxicology Webinar series
Slide 7
Introduction of exogenous bacteria to germ-free
mice: Expression of hepatic drug-processing genes
•
Age-matched 2-month-old adult conventional (CV) and
germ-free mice (males, n=5~8 per group) were
administered 4.5×106 CFU/ml drinking water for 28-days
•
In a separate experiment, 1-month old GF mice were
taken out of the isolator and housed with the feces from
CV mice for 2-months
•
All mice were 3-month-old prior tissue collection.
American College of Toxicology Webinar series
Slide 8
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Regulation of the Cyp3a Gene Cluster by VSL3 and
Conventionalization
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Selwyn, Cheng, Klaassen, and Cui JY*, 2016 Drug Metab Dispos, 44: 262-74
American College of Toxicology Webinar series
Slide 9
Regulation of the Cyp4a Gene Cluster by VSL3 and
Conventionalization
Selwyn, Cheng, Klaassen, and Cui JY*, 2016 Drug Metab Dispos, 44: 262-74
American College of Toxicology Webinar series
Slide 10
Validation of the Protein Expression of Cyp3a and
Cyp4a
Selwyn, Cheng, Klaassen, and Cui JY*, 2016 Drug Metab Dispos, 44: 262-74
American College of Toxicology Webinar series
Slide 11
Validation of Enzyme Activity of Cyp3a and Cyp4a
Cyp3a11
Cyp4a14
Selwyn, Cheng, Klaassen, and Cui JY*, 2016 Drug Metab Dispos, 44: 262-74
American College of Toxicology Webinar series
Slide 12
PXR- and PPARα-DNA Binding Sites in Control
Mouse Liver
PXR binding fold-enrichment to Cyp3a loci in mouse liver
A
Site 5
Site 4
Site 3
Site 2
Site 1
(Data re-analyzed from the PXR ChIP-Seq experiments in control
mouse liver, from a previous study by Cui et al., 2009)
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PPARα-binding to Cyp4a loci in liver
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ID+GSM1514929;+original+experiments+were+performed+by+Lee+et+al,+2014.)+
Selwyn, Cheng, Klaassen, and Cui JY*, 2016 Drug Metab Dispos, 44: 262-74
American College of Toxicology Webinar series
Slide 13
ChIP of PXR and PPARα binding to Cyp3a and Cyp4a
loci
Selwyn, Cheng, Klaassen, and Cui JY*, 2016 Drug Metab Dispos, 44: 262-74
American College of Toxicology Webinar series
Slide 14
Germ-free and conventionalization have the most prominent effect
on the regulation of hepatic drug-processing genes
• Germ-free conditions resulted in the most prominent
changes in hepatic drug-metabolizing enzyme
expression, most notably a consistent down-regulation
of many genes in the Cyp3a cluster, but a consistent upregulation of many genes in the Cyp4a cluster.
• Conventionalization of GF mice at least partially restores
the expression of these genes to CV levels.
•
The GF and conventionalization mediated changes in
Cyp3a and 4a genes are associated with altered PXR
and PPARα-binding to the targeted DNA sequences
within these genes.
American College of Toxicology Webinar series
Slide 15
Polybrominated Diphenyl Ethers (PBDEs)
• PBDEs are among the most abundant and persistent
environmental chemicals in the human population
BDE-47
(2,2’,4,4’-Tetrabromodiphenyl ether)
BDE-99
(2,2’,4,4’,5-Pentabromodiphenyl ether)
• BDE-47 and BDE-99 are the predominant congeners
detected in humans, and they are highly prevalent in
seafood and breast milk at worrisome levels humans
American College of Toxicology Webinar series
Slide 16
Hepatic Biotransformation of PBDEs and Toxicities
• Hepatic uptake: via organic anion transporting
polypeptides (OATP) transporters
• Hepatic Phase-I metabolism: by human CYP2B6 to toxic
hydroxylated metabolites
• Hepatic Phase-II conjugation (in vitro evidence):
glucuronidation, sulfonation or methylation
• Toxicities of PBDEs: thyroid toxicity, neurodevelopmental
disorders, hepatic oxidative stress and cancer.
American College of Toxicology Webinar series
Slide 17
BDE-47 is a CAR activator
Sueyoshi et al., 2014, Toxicol Sci, 137: 292-302
American College of Toxicology Webinar series
Slide 18
Sulfotransferase 5a1: important for Phase-II
Sulfonation conjugation reaction of xenobiotics
• Sult5a1 is up-regulated by the
CAR ligand TCPOBOP in
livers of wild-type mice
• The TCPOBOP-mediated upregulation of Sult5a1 is absent
in livers of CAR-null mice
• Sult5a1 is a bona fide CARtarget gene
• PBDE-47 mediated upregulation of Sult5a1 depends
on the presence of gut
microbiome.
Davis Cowles and Julia Cui
American College of Toxicology Webinar series
Slide 19
Experimental Design
•
CV and GF mice were treated with vehicle (corn oil),
BDE-47, or BDE-99 via oral gavage once daily for 4-days
•
GC-MS quantification of PBDE metabolites
•
RNA-Seq of hepatic transcriptiome
American College of Toxicology Webinar series
Slide 20
Hepatic Drug-processing Genes Related to PBDE
Biotransformation
American College of Toxicology Webinar series
Cindy Li and Julia Cui
Slide 21
PBDE-mediated regulation of the hepatic transcriptome
is profoundly modified by lack of gut microbiota
• Gut microbiota-dependent regulation
• Potentiation effect due to lack of gut microbiota
Control vs. BDE-47
GF
CV
966
116
754
Control vs. BDE-99
GF
CV
629
586
4302
Cindy Li and Julia Cui
American College of Toxicology Webinar series
Slide 22
Summary of PBDE Oxidative Metabolism in Mouse
Liver
Cindy Li and Julia Cui in Collaboration with Dr. Irvin Schultz
American College of Toxicology Webinar series
Slide 23
Overall Conclusion
• Gut microbiota regulates the expression and activity of
various drug-processing genes in liver
• PXR and PPARα appear to be the targets which the
intestinal bacteria interact with
• The presence of gut microbiota alters the hepatic
metabolism of PBDEs, and it is necessary in the PBDEmediated regulation of many hepatic genes involved in
drug metabolism and other critical endogenous
pathways
• Future studies will determine which specific bacterial
strains and their microbial metabolites interact with the
host receptors in liver
American College of Toxicology Webinar series
Slide 24
Looking into the Future:
Microbiome is a Key Player in Exposome
• Microbiome and Toxicology (mice as a research model)
• Microbiome and nutrition
• Microbiome and early life exposure/epigenetics
• Microbiome and “Big Data”
• Microbiome as biomarkers for risk assessment
• Probiotics as preventive therapy to reduce toxic
exposure in vulnerable populations
American College of Toxicology Webinar series
Slide 25
Acknowledgements
Members in the Cui Laboratory
Collaborators:
• Felcy Selwyn, former Research Scientist
• Dr. Bhagwat Prasad (UW)
• Dr. Irvin Schultz (PNNL)
• Sunny Cheng, Research Scientist
• SooWan Lee, Research Scientist
• Cindy Li, PhD Student
• Joseph Dempsey, PhD Student
• Gurkirat Sidhu, Undergraduate Student
• Daniel Park, Undergraduate Student
• Khakkhak Khayi, Undergraduate Student
• Felcy Selwyn, former Research Scientist
• Yubin Song, former Undergraduate Student
• Shinhee Park, former Undergraduate Student
• Elaine Chen, former Undergraduate Student
• Davis Houston Cowles, former EHREP Intern
Supported by National Institute of Health R01 grants GM111381, ES025708, and R01 ES019487, UW start-up funds,
and Murphy Endowment.
American College of Toxicology Webinar series
Slide 26
Aaron Ericsson, DVM, PhD
Dr. Ericsson received a DVM from the University of Missouri (MU) in 2006 and
went on to complete a residency in Laboratory Animal Medicine in 2009,
followed by a T32-funded PhD in Area Pathobiology, focusing on the role of the
innate immune system in gastrointestinal inflammation and colitis-associated
colorectal cancer (CAC).
In 2013, he assumed the role of lead scientist for microbiome research at both
the NIH-funded MU Mutant Mouse Resource and Research Center (MMRRC)
and Rat Resource and Research Center (RRRC), and received funding to found
the MU Metagenomics Center in 2014.
He is primarily funded by a K01 to investigate the influence of certain microbes
and polymicrobial communities on CAC, and also collaborates heavily with NIHand USDA-funded investigators.
American College of Toxicology Webinar series
Slide 27
Julia Yue Cui, PhD
Julia received her B.S. Degree in Chukechen Honors College,
Zhejiang University in Hangzhou, China, and received her Ph.D.
Degree with honors in University of Kansas Medical Center. Julia
currently is an Assistant Professor in Toxicology in the Department of
Environmental and Occupational Health Sciences. She is a recipient
of the Sheldon D. Murphy Endowed Chair, and a member of Center of
Ecogenetics & Environmental Health. Julia is trained as a toxicologist,
specializing in using toxicogenomic and toxicoepigenomic approaches
to determine the effects of environmental chemical exposure and
reprogramming the gut microbiome on the transcriptional and
epigenetic regulation of genes involved in drug metabolism and
obesity during development.
American College of Toxicology Webinar series
Slide 28
Thank you for your
participation in the
American College of Toxicology
Webinar!
We hope to see you at the
37th Annual Meeting of
the American College of Toxicology.
American College of Toxicology Webinar series
Slide 29