Reviewing QACs for Biomonitoring: Metabolism, …...2020/03/04 · Metabolism, Analytical...
Transcript of Reviewing QACs for Biomonitoring: Metabolism, …...2020/03/04 · Metabolism, Analytical...
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Reviewing QACs for Biomonitoring: Metabolism, Analytical Considerations, and Effects on Cholesterol Homeostasis
Libin Xu, PhD Assistant Professor
Department of Medicinal Chemistry University of Washington
03/04/2020
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Quaternary Ammonium Compounds
Disinfectants, pesticides, preservatives
Regularly used in a variety settings 1,2
Cleaning products, eye drops, utensils, laundry detergent, milking equipment 1,2
Dermal (eye), ingestion, inhalation exposure routes
1) Hrubec , T., et al ( 2017 ) Birth Defects Research 2) US EPA ( 2006 ) RED for ADBAC, EPA739-R-06-0092
(BACs)
(DDAC)
No Public Data On QAC Exposure Levels In Humans!
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Quantitation of QACs by Liquid Chromatography-Tandem Mass Spectrometry
Internal Standards:
d7-BACs
LC Conditions: Mobile phase: gradient of Solvent A (water, 0.1 % formic acid, 2 mM ammonium formate ) and Solvent B (acetonitrile)UPLC column: Thermo Hypersil GOLD C18 ( 100 x 2.1 mm, 1.9 mm) Run time: < 8 min
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Exposure Levels in 100 Random Human Plasma Samples
Random human plasma obtained from BioIVT
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Can Human Body Metabolize QACs?
Using benzalkonium chlorides (BACs) as examples.
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Metabolism by Human Liver Microsomes
BACs Half-Life(minutes)
C10 1C12 3C14 7C16 15
Metabolism is cofactor NADPH - dependent à cytochrome P450 involvement
Seguin, R. P., et al. (2019) Chem. Res. Toxicol., 32(12), 2466 – 2478.
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Screening Recombinant CYPs Identifies Responsible CYP Isoforms
C10-BAC% remaining
C16-BAC% remaining
CYPs 2D6, 4F2, and 4F12 are major metabolizing enzymes!
These enzymes are highly polymorphic in humans!
Seguin, R. P., et al. ( 2019 ) Chem. Res. Toxicol., 32( 12 ), 2466 – 2478 .
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What Metabolites are Formed from BACs?
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Primary products: ω - and (ω - 1) - hydroxylation
Rel
ativ
e A
bund
ance
ω-OH(ω-1)-OH
ω-OH ω-OH ω-OH
(ω-1)-OH
(ω-1)-OH (ω-1)-
OH
Seguin, R. P., et al. (2019) Chem. Res. Toxicol., 32(12), 2466 – 2478.
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Primary Products Undergo Further Metabolism
Seguin, R. P., et al. (2019) Chem. Res. Toxicol., 32(12), 2466 – 2478.
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Metabolites Can Be Quantified by LC-MS/MS
Metabolites of C10-BAC
Seguin, R. P., et al. ( 2019 ) Chem. Res. Toxicol., 32( 12 ), 2466 – 2478 .
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DDAC
C16-BACC14-BAC
C12-BAC
QAC-fed kidney tissue
Control kidney tissue
m/z filtered:304.300 (C12-BAC)332.332 (C14-BAC)326.379 (DDAC)360.363 (C16-BAC)
QACs Detected in Kidney Tissue from Mice Exposed to QACs Via Diet
120 mg/kg/day QAC (40%BAC and 60%DDAC) for two weeks following Melin et al. Reprod. Toxicol. 2014.
Seguin, R. P., et al. (2019) Chem. Res. Toxicol., 32(12), 2466 – 2478.
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QAC Metabolites are Observed in Mice Fed a QAC Diet
Control kidney tissue
QAC - fed kidney tissue
HLM Metabolites
Metabolites of BAC - C 14 and C 16 are also observed!
Ryan Seguin
Intensity: 367
Intensity: 1.51e4
Intensity: 1.26e6
C12-BAC metabolites
ω-OH(ω-1)-OH
ω-OH(ω-1)-OH
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Effect of Benzalkonium Chlorides on Cholesterol and Lipid Homeostasis: Why Are We Interested in It?
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Why Do We Study Benzalkonium Chlorides (BACs)?
Smith-Lemli-Opitz syndrome (SLOS) Mutations
Inhibition by drugs or environmental moleculesNeurodevelopmental
Defects
• 1 in 10,000 to 60,000
• Congenital malformation, mental
retardation, autistic behavior
• Carrier frequency: 1 in 30
Small molecule inhibitors of DHCR 7 :
• Pharmaceuticals: breast cancer, antipsychotics
• Disinfectants: benzalkonium chloridesPorter, F. D.; Herman, G. E. J. Lipid. Res. 2011, 52, 6 .
Canfran - Duque et al. J. Lipid Res. 2013
Korade , Z. et al. J. Med. Chem. 2016
Herron, J. et al. Tox. Sci. 2016, 151, 261.
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Benzalkonium Chlorides Are Structurally Similar to Known Inhibitor of DHCR7, AY9944
“%” indicates similarity score.
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Benzalkonium Chlorides Inhibit DHCR7 in a Chain Length-Dependent Manner
Herron et al. Tox. Sci. 2016
Neuro2a cells were exposed to each compound at 100 nM for 2 days
Known inhibitor BAC: highest similarity to AY9944 (72.9%) among molecules in Tox21
*, p < 0.05**, p < 0.005***, p < 0.0005
***
*****
7-Dehydrocholesterol
**
Cholesterol
********
******
Desmosterol
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BACs Alter Lipid Homeostasis in Neuronal Cells
Herron, J. et al. ( 2016 ) Toxicol . Sci. Hines, Herron, and Xu. ( 2017 ) J. Lipid Res. 18
Nor
mal
ized
Abu
ndan
ce (x
104 )
Control AY9944 C10 C162.0
1.5
1.0
0.5
0.0-500
-400
-300
-200
-100
0
100
200
300
400
500
-800 -600 -400 -200 0 200 400 600 800
Prin
cipa
l Com
pone
nt 2
(22.
6%)
Principal Component 1 (59.8%)
ControlAY9944C10C16
A 7-DHC metabolite
Neuro2a cells were exposed to each compound at 100 nM for 2 days
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Individual BACs Alter Gene Expression Involved in Sterol and Lipid Homeostasis
Herron, J. et al. (2016) Toxicol . Sci. Hines, Herron, and Xu. (2017) J. Lipid Res.
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*** ** ** **
*** *** *** ** **** ** **
* *
Cholesterol synthesisCholesterol regulation
Fatty acid synthesisCholesterol efflux
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Can BACs Alter Sterol and Lipid Homeostasis In Developmental Brain?
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In utero Exposure to BACs – Experimental Design
Herron, et al. (2019) Toxicol. Sci. 21
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BACs Cross the Placenta and Enter the Developing Brain
Herron, et al. ( 2019 ) Toxicol. Sci. 22
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BACs Decrease Sterols in Neonatal Brains
23Herron, et al. (2019) Toxicol. Sci.
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BACs Alter Lipidome of Neonatal Brains
Herron, et al. (2019) Toxicol. Sci.
Triglycerides
Diglycerides
Hexosylceramides
Ceramides
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What Underlying Gene Expression Changes Are Associated with Alterations in Sterols and Lipids? RNA Sequencing
25
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Global Gene Expression Changes in Exposed Neonatal Brains
Herron, et al. (2019) Toxicol. Sci. 26
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Cholesterol Biosynthesis Pathway is Activated in Exposed Neonatal Brains
Herron, et al. ( 2019 ) Toxicol. Sci. 27
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Upstream regulator SREBP cleavage - activating protein (SCAP) is activated in exposed neonatal brains
Herron, et al. (2019) Toxicol. Sci. 28
BAC C12 Adjusted P-value = 2.02E-12
BAC C16 Adjusted P-value = 4.37E-07
Cholesterol synthesis
Cholesterol regulation
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Regulation of Cholesterol Homeostasis by Scap and Insig
Goldstein, J. L., et al. (2006). Cell, 124(1), 35 – 46.
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Distinct Expression Patterns of Genes Involved in Sterol - And Lipid- Homeostasis
Herron, et al. (2019) Toxicol. Sci. 30
Cholesterol biosynthesis genes
Insulin induced gene 1 (Insig1)
Low density lipoprotein receptor
ELOVL fatty acid elongase 6
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Distinct Expression Patterns of Genes Involved in Sterol - And Lipid- Homeostasis
Herron, et al. (2019) Toxicol. Sci. 31
Apolipoprotein A1/C1/A2
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Summary• QACs are observed in random human plasma samples. • BACs can be metabolized by human cytochromes P 450 . • BACs and metabolites can be quantified by LC - MS/MS and
both should be used for biomonitoring for complete assessment of BAC exposure.
• BAC exposure leads to elevated levels of parent compounds and metabolites in dam and neonatal tissues.
• BACs disrupt cholesterol and lipid homeostasis both in vitro and in vivo.
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Acknowledgements
• Colleagues in Dept. of Med. Chem. and School of Pharmacy
• School of Pharmacy Mass Spectrometry Center (Dale Whittington, Scott Edgar)
• Funding: NIH (R01HD092659, R01AI136979), SOP Innovation Fund, UW Royalty Research Fund
– Josi Herron, Libin, Amy Li, Quynh Do, Emily Pruitt, Vanessa Lopez, Jonathan Palmer, Ryan Seguin, Dylan Ross, Rutan Zhang
Kelly Hines (now at UGA Chemistry)
Hideaki Tomita