1. Inhibition of a High Fidelity DNA Repair Pathway by the Environmental Pollutant Hexavalent Chromium
Cynthia Browning
Wise Laboratory of Environmental and Genetic Toxicology
University of Louisville, KY
Graduate School of Biomedical Science and Engineering
University of Maine, ME
GSBSE Annual Meeting
September 16, 2016

2. Chromium (Cr) Naturally occurring metal Primary uses Exposure Hardness
Naturally occurring metal
Hardness
High resistance to corrosion
Cr salts have bright colors
Primary uses
Corrosion resistant agent
Pigment in dyes, inks and paints
Exposure
Occupational
Environmental
ium2hpD9m_A/s1600/B25-mitchell-assembly.jpg
Use as a primer to resist corrosion started in the 1920s by the Ford Motor company.
Resistance to corrosion makes it popular for primers, chrome plating and as a stainless steel alloy.
Sources of exposure to chromium: Paints, Dyes, Inks and Their Dusts
Combustion of Fossil Fuels
Industrial Cooling Towers
Catalytic Converter Exhaust
Trash Incinerator Exhaust
Cement Dust
Brake Lining Dust
Industrial Waste
Leather Materials
Cigarette Smoke

3. Chromium (VI) Carcinogenicity
Particulate Cr(VI) is a human lung carcinogen
Cr-induced tumors are characterized by genomic instability
Particulate Cr(VI) induces chromosome instability
Homologous recombination
repair protects against
Cr(VI)-induced structural
chromosome instability
Cr(VI)
Epidemiological studies have identified chromate-induced lung cancers in chromate workers, chromium platers and dyers. Chromate is suspected to contribute to lung cancer formation in electroplaters, welders, metal workers, glass-fiber manufacturers and tanners.
From epi studies
From pathology studies and animal studies
Other health effects include: skin irritation and ulceration; decreased sperm counts; kidney and liver damage; immunosuprression

4. Effect of Cr(VI) Exposure on Homologous Recombination (HR) Repair
Normal Cells
24 h Cr(VI) Treatment
> 72 h Cr(VI) Treatment
DSB
DSB
DSB
MRE11
Sensing
MRE11 M N R
Sensing
Sensing M N R M N R M N R M N R
p-ATM M N R
Transducing
p-ATM M N R
Transducing
p-ATM
p-ATM M N R
Transducing
p-ATM
Nuclear RAD51 M N R
p-ATM
Rad51
Effecting M N R
p-ATM
Rad51
Effecting M N R
Effecting
Nuclear RAD51
RPA
HR active
HR inhibited

5. Hypothesis
Prolonged Exposure to Particulate Cr(VI) Inhibits Homologous Recombination Repair Through the Misregulation of RAD51

6. Prolonged Cr(VI) Exposure Decreases RAD51 Protein Levels
Let’s start by examining the effect of Cr(VI) exposure on Rad51 protein levels
n=3

7. Prolonged Cr(VI) Exposure Inhibits RAD51 Nuclear Monofilament Formation

8. RAD51 Foci Formation Is Inhibited By Prolonged Cr(VI) Exposure

9. Prolonged Cr(VI) Exposure Induces Cytoplasmic Accumulation of RAD51
120h
0.2 ug/cm2
Zinc Chromate
24h
0.2 ug/cm2
Zinc Chromate

10. Prolonged Cr(VI) Exposure Increases Cytoplasmic RAD51

11. Nuclear Transport
RAD51 nuclear transport is tightly regulated to limit levels within the nucleus
Excessive nuclear levels of RAD51 can induce random recombination events
RAD51 nuclear import facilitated by
RAD51C
BRCA2
RAD51 nuclear export facilitated by
CRM1
Directionality of nuclear transport
Ran-GTP predominantly found in nucleus
Promotes dissociation of importin/cargo to release cargo in nucleus
RanGAP1 is located on the cytoplasmic side of the NPC and stimulates GTP hydrolysis
Stabilizes CRM1/cargo complexes in nucleus
Ran transported back into the nucleus
Ran-GDP predominantly found in cytoplasm
Export cargo is released in the cytoplasm
RCC1 facilitates GTP loading of Ran in the nucleus so the cycle can repeat

12. Prolonged Cr(VI) Exposure Inhibits RAD51C Foci Formation
However, if we look at function by the formation of RAD51C foci, we find that RAD51C is inhibited after prolonged Cr(VI) exposure.
How is RAD51C being inhibited? Is it being prohibited from entering the nucleus?

13. Prolonged Cr(VI) Exposure Increases Cytoplasmic RAD51C

14. RAD51 Cytoplasmic Accumulation Increases After Cr(VI) Exposure in RAD51C Deficient Cells
Based on threshold set on the average cytoplasmic fluorescence of wt control
n=3

15. Prolonged Cr(VI) Exposure Slightly Increases Cytoplasmic BRCA2

16. Prolonged Cr(VI) Exposure Inhibits the Central HR Repair Protein RAD51
RAD51 functional unit is inhibited
Decreased protein level
Subcellular mislocalization
RAD51 import is inhibited
RAD51C and BRCA2 are sequestered in the cytoplasm
RAD51C foci formation inhibited
RAD51C deficiency induces RAD51 cytoplasmic accumulation
Suggests the RAD51 cytoplasmic accumulation is due to impaired import. May have more widespread consequences if nuclear import itself is inhibited.
Does prolonged Cr(VI) exposure inhibit homologous recombination repair?

17. Prolonged Cr(VI) Exposure Inhibits Homology Directed Repair
No Homology Directed Repair
n=3

18. Prolonged Cr(VI) Exposure Inhibits Sister Chromatid Exchange Formation

19. Proposed Mechanism of Particulate Cr(VI)-Induced Carcinogenesis
Lower Fidelity DNA Repair Pathways G2
Arrest
HR Repair Deficiency
Cation
DNA Double Strand Breaks
RAD51 inhibition
Cr 3+
Cr 5+ O
Cr 4+
Structural Chromosome Instability
Cr 6+
Decreased RAD51 Protein Level
Subcellular Mislocalization of RAD51 Protein
Anion Transport
Permanent and Heritable
Cell Phenotype Change
Misregulation of RAD51 Nuclear Import
Neoplastic Transformation and Cancer
Cell Membrane

20. Acknowledgements
Wise Lab of Environmental and Genetic Toxicology, University of Louisville
Dr. John Pierce Wise, Sr
Dr. Sandra Wise
Christy Gianios
Rachel Speer
Sean Raph
James Rossman
Committee Members
Dr. Hong Xie, Dr. Doug Thompson, Dr. Kevin Mills,
Dr. Chris States
Graduate School of Biomedical Science and
Engineering, University of Maine