KERATINOCYTE DIFFERENTIATION AND TOXIC TARGET BEHAVIOR

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010510
• Project No.: CA-D-ETX-2152-H
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2021

Project Director
Rice, R.

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Environmental Toxicology

Non Technical Summary
The maturation of epidermis produces a callus layer at the skin surface that provides our major protection against toxic agents in the environment. If epidermal cells do not mature properly, we are not protected well against environmental chemicals, and certain skin diseases may result even without chemical exposure. Studying how epidermal cells respond to toxic exposures can help us understand how cell functions can be perturbed and how disease states can result. The proposed work will compare antimony (about which relatively little is known) with arsenic, a known carcinogen that it resembles chemically. The results could indicate whether further investigation of the possible human carcinogenicity of antimony is warranted. In addition, the mechanism by which the notorious pollutant TCDD (dioxin) perturbs epidermal cells will be studied. The results could help understand how chemicals that act in the same signaling pathway influence cell behavior and how other genes can modify the response. For example, in certain skin diseases it is known that a given gene is defective, but it is not known why individuals with the same defect can differ so dramatically in severity of symptoms. Surprisingly, liquid smoke extract produces some of the same effects in epidermal cells as does TCDD. Studying the response to this common food flavoring ingredient, a form of concentrated air pollution available for purchase at the supermarket, may help understand some health effects of exposure to forest fires. Finally, copper is commonly used as a pesticide to suppress fungal growth and, in the marine environment, degradation of wooden structures, but the molecular mechanisms are poorly understood. However, comparison with certain effects of arsenic suggests a signaling pathway that may be operative. Finally, we plan to continue examining the protein content of epidermal derivatives, particularly hair shaft, as a source of information about natural fibers, individual identity and possibly disease state and effects of environmental chemical exposure.

Animal Health Component

0%

Research Effort Categories

Basic

80%

Applied

10%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
711	7010	1150	25%
314	5220	1160	25%
723	3840	1030	25%
308	3699	1040	25%

Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals; 308 - Improved Animal Products (Before Harvest); 723 - Hazards to Human Health and Safety;

Subject Of Investigation
7010 - Biological Cell Systems; 3840 - Laboratory animals; 3699 - Sheep and wool, general/other; 5220 - Pesticides;

Field Of Science
1040 - Molecular biology; 1160 - Pathology; 1030 - Cellular biology; 1150 - Toxicology;

Keywords

antimony

arsenic

cell culture

copper

corneocyte

hair

keratinocyte

liquid smoke

mass spectrometry

proteomics

tcdd

Goals / Objectives
Our research focuses on the keratinocyte, the major cell type in epidermis and other stratified squamous epithelia in humans and other mammals. This cell type is appropriate for understanding toxic mechanisms, since it forms the primary extra-pulmonary barrier to our environment, and thus is a frequent target of chemical agents, and it is one of the few epithelial cell types that is amenable to serial culture. The responses of keratinocytes are often good indicators of those in other epithelial cell types. In this direction, we plan to examine responses of the cells to four agents of concern. First, we will examine the mechanism by which inorganic arsenic alters epidermal cell function, eventually leading to cancer of the skin and internal organs. We anticipate doing so in parallel to inorganic antimony, which appears to have similar chemical properties but is not yet recognized as a human carcinogen. Second, we will continue studying the action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a known animal carcinogen and tumor promoter and human chloracnegen. Its mobilization of intracellular calcium and the influence of modifier genes will be examined. Third, the toxic effects of the common food flavorant liquid smoke will be studied, known to be toxic to micro-organisms. We have found that it activates the aryl hydrocarbon receptor and appears to increase intracellular calcium levels as does TCDD. Finally, we will investigate the effect of copper ion on keratinocyte function. This is widely used as a pesticide in the marine environment and as a fungicide in orchards, but its mechanism of toxicity is poorly understood. In addition to studying keratinocyte responses to various toxic chemicals, we also plan to continue developing methods to identify individuals and species by their hair proteomes with possible applications to forensic investigation, fiber improvement, assessing environmental exposure and diagnosis of medical afflictions.

Project Methods
Mechanisms of toxic action will be explored in keratinocytes cultured primarily from human epidermis. At our disposal are normal keratinocytes and a keratinocyte line that became immortalized spontaneously (SIK). In most cases, experiments are performed first using the SIK line for convenience and then repeated in normal strains to demonstrate the degree to which the observed phenomenon is also observed. For this purpose, the cells are cultured using a feeder layer of lethally irradiated mouse 3T3 fibroblasts, which provides growth factors necessary for serial passaging, in standard cell culture medium supplemented with serum, hydrocortisone and epidermal growth factor. For studies of differentiation, the cells are exposed to the agent of interest (arsenic, antimony, copper, TCDD, liquid smoke) starting shortly before confluence, and the cells are maintained in this condition for approximately 10 days. The cultures are harvested and analyzed for growth potential (colony forming ability) and differentiation marker levels (immunoblotting, real time PCR). For proteomic analysis, samples of hair shaft (or sometimes other tissue material) are reduced with dithioerythritol in the presence of sodium dodecyl sulfate . The proteins treated this way are alkylated with iodoacetamide to block cysteine residues. The detergent is removed by precipitation or solvent extraction and then the protein is digested exhaustively with TPCK-treated trypsin stabilized by reductive methylation. The digest is then provided to the Proteomics Facility Core in the UCD Genome Center, where it is analyzed by mass spectrometry after reverse phase high performance liquid chromatography. Peptide masses are matched to a peptide database and reported as a Scaffold file summarizing peptide data and showing protein sequence coverage. The profile of identified proteins is ordinarily expressed as weighted spectral counts (discounting those proteins lacking exclusive counts). This approach permits comparison of samples analyzed in parallel such as those from humans or animals (or their cells) to investigate individual identity, protein profile, disease processes or consequences of environmental exposure.

Progress 10/01/16 to 09/30/21

Outputs
Target Audience:The target audiences we have reached include primarily scientists and students, but we anticipate reaching government and industry scientists as the new projects proceed. The latter two groups have incorporated our findings into their approach to proteomic analysis of hair and epidermis. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training for postdoctoral, PhD, MS and undergraduate students. How have the results been disseminated to communities of interest?Our research has been disseminated to the scientific community though publications in scientific journals, academic seminars and presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to pursue further mechanistic investigation of air pollutants (e.g., wood smoke and other combustion products) using human epidermal cells and a proteomic approach. In addition, we have derived a sturgeon epithelial cell line that we plan to use as a model for studying the response of such species to relevant pollutants, including those activating the aryl hydrocarbon receptor and stimulating metabolic activities.

Impacts
What was accomplished under these goals? One of the goals, to understand the mechanism of trivalent arsenic (and antimony) action in human epidermal cells, has resulted in a detailed transcriptional analysis. This led to identification of several unexpected upstream intracellular signaling regulators and to the recognition that trivalent antimony effects are virtually identical to those of arsenic. Thus we propose that antimony should be regarded as a human skin carcinogen. In the proteomic arena, we established that protein expression levels in hair are primarily genetically determined (on the basis of samples from monozygotic twins) and that sufficient genetically variant peptides are identifiable to form a basis for individualization. The statistical basis for this method is increasingly approaching the gold standard of nuclear DNA analysis. Similarly, this approach offers an additional use for fingerprints, which are amenable to the same proteomic analysis. Meanwhile, the protein profiling approach has permitted characterization of aberrant epidermis and hair from human disease states and from such states (primarily due to gene ablation) in animal models. Our latest work has shown that hair shaft samples are sufficient to establish genetic relationships in families. This will facilitate elucidating close relatedness of family membership that is in dispute, identify cases of hair disease and possibly the influence of environmental pollution. In another approach to the last goal, we have shown that exposure of human epidermal cells to wood smoke extract has readily detectable effects on their behavior in several respects. Cells growth is inhibited and, at lethal concentration, the cells synthesize cross-linked envelopes. Concentration-dependent stimulation is seen of expression of at least several genes indicating increased levels of xenobiotic processing, reactive oxygen and inflammation.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Karim N, Plott TJ, Durbin-Johnson BP, Rocke DM, Salemi M, Phinney BS, Goecker ZC, Pieterse MJM, Parker GJ, Rice RH (2021) Elucidation of familial relationships using hair shaft proteomics. For Sci Int Gen 54:102564
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Lin L-W, Denison MS, Rice RH (2021) Woodsmoke extracts cross-link proteins and induce cornified envelope formation without stimulating keratinocyte terminal differentiation. Toxicol Sci 183:128-138

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audiences we have reached include primarily scientists and students, but we have also reached government and industry scientists. The latter two groups have incorporated our findings into their approach to proteomic analysis of hair and epidermis. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provides training for postdoctoral, PhD, MS and undergraduate students. How have the results been disseminated to communities of interest?Our research has been disseminated to the scientific community though publications in scientific journals, academic seminars and presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Since we have largely completed the first aim of the original project, we plan to focus on the remaining aims, mostly the effects of liquid smoke/air pollution chemicals and TCDD on keratinocyte function. We plan to examine the effects of exposure of each on a critical feature of the cellular differentiation program that results in cell death. We plan to find how exposure affects the protein composition of the terminated cells promoted by both types of exposure, and then examine effects of specific chemical components of wood smoke. This plan will be to employ improvements in sample processing that we have developed so far.

Impacts
What was accomplished under these goals? This project has been directed to understanding effects of toxic chemicals on epidermal cells. While these are initially mostly from human, the results usually apply to farm animals and the environment more generally. We have focused on chemicals known or suspected of causing deleterious effects. Thus, arsenic is a known human skin carcinogen, and TCDD is known to cause a long lasting acne of the human skin. By contrast, liquid smoke flavoring (available in supermarkets) is quite toxic to micro-organisms, but its effect on human epithelia (including epidermis) is not well understood. This flavoring is a model for water-soluble components of air pollution, which are known to be deleterious for livestock and crops. Copper compounds are widely used as anti-fungal agents, but why fungi are so more sensitive than humans, animals or the crops and seeds being protected by it is unknown. To help analyze toxic effects on epidermis, we are studying its proteome so that we can compare how the proteins in it (and in appendages, including the hair shaft) are perturbed by chemical exposure. This approach, initially on human samples, can be readily translated to other species, including livestock and plant crops once the technical procedures are established. [1] In this project period, we reported that arsenic and antimony induce virtually identical transcriptional effects in human epidermal cells. This strengthens the case that antimony is probably a human skin carcinogen like arsenic, a finding that may lead to re-evaluation of its use as a flame retardant (increasing in prevalence), occurrence as a contaminant of food and its presence as a contaminant in soil and watersheds as a result of mining activity (change in action). [2,3] We have found that TCDD and liquid smoke flavoring both induce epidermal cell death. While TCDD appears to accelerate a normal physiological process, we have now found that liquid smoke appears to act differently, by overt toxicity (change in knowledge). [4] Since the other topics have kept us fully engaged, we have not investigated the action of copper. [5] We have focused instead on developing proteomic analyses of epidermis and hair by improving processing procedures for greater efficiency and characterizing effects of age and pigmentation status (change in action). We anticipate being able to incorporate these advances in analysis of toxic effects, medical and agricultural diagnostics and individual identification.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Parker G, Goecker Z, Franklin R, Durbin-Johnson B, Milan J, Kareem N, De Elon C, Matzoll A, Borja T, Rice B (2019) Proteomic genotyping: Using mass spectrometry to infer SNP genotypes in a forensic context. For Sci Intl: Genet Supp Ser 7:664-666
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Phillips MA, C�novas A, Islas-Trejo A, Medrano JF, Rice RH (2020) Deducing signaling pathways from parallel actions of arsenite and antimonite in human epidermal keratinocytes. Sci Rep 10:2890
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Eckhart L, Lachner J, Tschachler E, Rice RH (2020) TINCR is not a noncoding RNA but encodes a protein component of cornified epidermal keratinocytes. Exp Dermatol 29:376-379
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Franklin RN, Karim N, Goecker ZC, Durbin-Johnson BP, Rice RH, Parker GJ (2020) Proteomic genotyping: Using mass spectrometry to infer SNP genotypes in pigmented and non-pigmented hair. Forensic Sci Int 310:110200
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Plott TJ, Karim N, Durbin-Johnson BP, Swift DP, Youngquist RS, Salemi M, Phinney BS, Rocke DM, Davis MG, Parker GJ, Rice RH (2020) Age-related changes in hair shaft protein profiling and genetically variant peptides. For Sci Int Gen 47:102309
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Goecker ZC, Salemi MR, Karim N, Phinney BS, Rice RH, Parker GJ (2020) Optimal processing for proteomic genotyping of single human hairs. For Sci Int Gen 47:102314
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Hong BV, Lee JH, Rice RH (2020) Off-target effects of protein tyrosine phosphatase inhibitors on oncostatin M-treated human epidermal keratinocytes: the phosphatase targeting STAT1 remains unknown. PeerJ 8:e9504

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Target audience includes primarily scientists and students and may include government and industry scientists and regulators. We will also be alert to interactions with stakeholders affected by toxic exposures. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We continue to offer training for postdoctoral, PhD, MS and undergraduate students. How have the results been disseminated to communities of interest?Our research has been disseminated to the scientific community though publications in scientific journals and presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Plans for coming year include further investigation of (1) aryl hydrocarbon receptor involvement in epidermal terminal differentiation, (2) interaction of chemicals in wood smoke with epidermal cell signaling pathways and (3) development of a fish epithelial cell culture model.

Impacts
What was accomplished under these goals? We have contributed to development of a method for personal identification through proteomic analysis of hair and fingerprints. This method provides a major increase in the value of hair and fingerprint evidence.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Milan J, Wu P-W, Salemi M, Durbin-Johnson B, Rocke DM, Phinney BS, Rice RH, Parker GJ (2019) Comparison of protein expression levels and proteomically-inferred genotypes using human hair from different body sites. For Sci Intl: Genet 41:19-23
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Karim N, Durbin-Johnson B, Rocke DM, Salemi M, Phinney BS, Naeem M, Rice RH (2019) Proteomic manifestations of genetic defects in autosomal recessive congenital ichthyosis. J Proteomics 201:104-109
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Karim N, Phinney BS, Salemi M, Wu P-W, Naeem M, Rice RH (2019) Human corneocyte proteomics reveals cross-linking of a broad spectrum of proteins in cornified envelopes. Exp Dermatol 28:618-622
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Borja T, Karim N, Goecker Z, Salemi M, Phinney BS, Naeem M, Rice RH, Parker GJ (2019) Proteomic genotyping of fingermark donors with genetically variant peptides. For Sci Int: Genet 42:21-30
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Chan LK, Nguyen KQ, Karim N, Yang Y, Rice RH, He G, Denison MS, Nguyen TB (2019) Relationship between the molecular composition, visible light absorption, and health-related properties of smoldering woodsmoke aerosols. Atmospheric Chemistry and Physics, in press

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Target audience includes primarily scientists and students and may include government and industry scientists and regulators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have been training several PhD, MS and undergraduate students. How have the results been disseminated to communities of interest?Our research has been disseminated to the scientific community though publication in scientific journals. What do you plan to do during the next reporting period to accomplish the goals?We are focusing on proteomic analysis of epidermis and adnexa. We are also investigating signaling pathways perturbed in keratinocytes by arsenic and antimony.

Impacts
What was accomplished under these goals? We have established procedures for proteomic analysis of epidermis and adnexa and made numerous findings about the differentiation process occurring in their proteomes. We have also extended the work to epidermal (sebaceous gland) lipids.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Rice RH, Durbin-Johnson BP, Mann SM, Salemi M, Urayama S, Rocke DM, Phinney BS, Sundberg JP (2018) Corneocyte proteomics: Applications to skin biology and dermatology. Exp Dermatol 27(8):931-938
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Sundberg JP, Shen T, Fiehn O, Rice RH, Silva KA, Kennedy VE, Gott NE, Bechtold L, Murray S, Kuiper R, Pratt CH (2018) Sebaceous gland abnormalities in fatty acyl CoA reductase 2 (Far2) null mice result in primary cicatricial alopecia. PLoS One 13(10):e0205775
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Jaeger K, Sukseree S, Zhong S, Phinney BS, Mlitz M, Buchberger M, Narzt MS, Gruber F, Tschachler E, Rice RH, Eckhart L (2019) Keratinocyte cornification requires autophagy for bulk degradation of intracellular proteins while sparing components of the cytoskeleton. Apoptosis https://doi.org/10.1007/s10495-018-1505-4

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Target audiences include primarily research workers (scientists and students). Depending on the results, the audience could include government and industry scientists and regulators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have provided opportunities for 4 MS degree students and 2 PhD degree students to learn laboratory techniques that will equip them to perform research industry or academia. This background would also help such students enter careers in environmental health or related administration and regulatory positions. How have the results been disseminated to communities of interest?In addition to publishing the results of our studies, the students and I have participated in scientific meetings. In addition, I gave two lectures to (and led discussion among) an audience of interested citizens in a nearby town on the topic of toxic tragedies that occur through occupational, consumer and environmental contamination. What do you plan to do during the next reporting period to accomplish the goals?We are busily engaged in conducting studies on effects of arsenic, antimony, dioxin and wood smoke constituents on the health of human epidermal cells in culture. The information to be gained will be of importance in elucidating toxic mechanisms and thus the risk of exposures to such chemical agents.

Impacts
What was accomplished under these goals? We have discovered that the proteome of the human hair shaft is determined primarily by a person's genome. Using identical twins we found that the protein profiles of hair shafts from two members of a twin pair was nearly identical, whereas hair shafts from individuals in different twin pairs were easily distinguished by protein profiling. The influence of environment appeared minimal. This finding indicates that a pathway to improving the quality of natural fiber (e.g., wool) should focus on breeding. We have found that individuals suffering from the rare skin disease pachyonychia congenita have markedly altered protein profiles in regions of the foot epidermis that are affected. The disease results from a mutation in a gene encoding an important keratin expressed in the epidermis, but the expression of many other genes is affected. This information is anticipated to help monitor the value of treatments beyond the information obtained by visual inspection alone. It also may provide a method to evaluate effects of skin exposure to toxic substances in the environment. We have uncovered a step in the evolution of epidermis by terrestrial animals such as mammals using fish. We hypothesized that a critical enzyme expressed in our skin was present in the skin of fish and was adapted to making a structure critical for our epidermis. This structure evolved to provide a scaffold for attaching lipids to prevent desiccation of the skin. Indeed, we found that the enzyme is question, whose role in fish is not known, was present in many orders of bony fish. In performing this work, we developed a method to culture fish epithelial cells that may be of value for evaluating toxic exposures to aquatic species. While evaluating the response of human epidermal cells to vanadate, a common environmental contaminant, especially of air pollution, we observed that it strongly stimulated the cellular response to a certain class of hormones secreted by white blood cells (cytokines). This finding helps uncover a potential environmental exposure that could exacerbate responses to other pollutants.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Rice RH, Durbin-Johnson BP, Salemi M, Schwartz ME, Rocke DM, Phinney BS (2017) Proteomic profiling of Pachyonychia congenita plantar callus. J Proteomics 165: 132 -137
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wu P-W, Mason KE, Durbin-Johnson BP, Salemi M, Phinney BS, Rocke DM, Parker GJ, Rice RH (2017)Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides. Proteomics 17, 13 -14, 2017, 1600462
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Rodriguez Cruz SI, Phillips MA, K�ltz D, Rice RH (2017) Tgm1-like transglutaminases in Tilapia (Oreochromis mossambicus). PLoS One 12(5): e0177016
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Hill T, Rice RH (2018) DUOX expression in human keratinocytes and bronchial epithelial cells: Influence of vanadate. Toxicol in Vitro 46:257-264