The research program in my laboratory is aimed at understanding the role of oxidative stress and inflammation in human disease, in particular atherosclerosis (hardening and thickening of arterial walls leading to heart attacks and strokes), and the ameliorating effects of micronutrients and dietary supplements.
One of the earliest events in atherosclerosis is dysfunction of the endothelium (a single cell layer lining arterial walls), leading to increased expression of cellular adhesion molecules (CAM) and monocyte chemoattractant protein-1 (MCP-1) by these cells. As a consequence, white blood cells called monocytes are recruited to the arterial wall, where they become resident macrophages and initiate chronic inflammation and atherosclerotic lesion development. We are performing biochemical, cell biological, animal, and human studies to investigate 1) the mechanisms and consequences of endothelial dysfunction, 2) the role of pro-oxidant transition metals like iron and copper in this process, and 3) the effectiveness of antioxidant and anti-inflammatory dietary compounds in ameliorating endothelial dysfunction.
We have found that lipoic acid and certain metal chelators (metal-binding agents) effectively inhibit CAM and MCP-1 expression by human aortic endothelial cells and suppress systemic as well as vascular inflammation and atherosclerosis in experimental mice. We are now performing two randomized controlled trials to investigate the effects of lipoic acid supplementation on oxidative stress, inflammation, serum triglycerides and lipoprotein profile, and other coronary risk factors in overweight or obese human subjects and patients with coronary artery disease. Additional projects in the laboratory investigate the biological mechanisms and metabolic effects of polyphenolic flavonoids and the interactions of lipoic acid and ascorbic acid (vitamin C) in improving vitamin C body status and antioxidant defenses in humans.
Our laboratory investigates vitamin E: why do we need it, how much do we need, what is the best way to consume it, and are there adverse effects from consuming too much?
Although plants synthesize eight similar molecules with vitamin E antioxidant activity, the only form used by humans is alpha-tocopherol. Nearly 100 years after vitamin E’s discovery, we recognize that alpha-tocopherol is required for human life, functions as a potent fat-soluble antioxidant, and is well regulated by the human body. However, because no specific biochemical pathway or enzyme has been identified that requires alpha-tocopherol as an essential component, it has been difficult to quantify the amount of vitamin E that is needed daily.
To understand the functions of alpha-tocopherol at the molecular level, several tools are available. We use various plant forms of vitamin E to test specific regulatory functions. The alpha-tocopherol transfer protein (TTP) in the liver is critical in this regard. We have developed a diet for zebrafish to make them vitamin E deficient. This “aquarium fish” is used by many toxicologists to study environmental toxins because the genes in the zebrafish are similar to many of those in humans. Using the zebrafish model, we are now seeking to better define the biological role of alpha-tocopherol.
We have studied how vitamin E metabolism serves to prevent excess accumulation of vitamin E in the body. We are currently investigating how these mechanisms might have adverse effects with respect to vitamin K metabolism.
Overall, the assessment of the delivery and function of vitamin E in humans has lagged because previously we lacked the appropriate tools. We are in the process of developing an intravenous preparation of vitamin E labeled with deuterium to be able, for the first time, to measure vitamin E absorption, biokinetics, and bioavailability of vitamin E in humans.
Our research seeks to identify the mode of action of two "age-essential" micronutrients, lipoic acid (LA) and acetyl-L-carnitine (ALCAR). This work is aligned with Dr. Pauling's concept of "orthomolecular medicine"—varying the concentrations of substances normally present in the body to affect health. We are using LA and ALCAR as "keys" to unlock important mechanisms associated with the basic biology of aging, which may lead to effective therapies for a number of age-related diseases and enhance the quality of life.
We found that ALCAR and LA improve two of the most important cellular lesions of aging: the inability to respond to oxidative and toxicological challenges and the loss of mitochondrial function. Feeding old rats LA markedly elevates both cellular ascorbic acid and glutathione levels, and induces Phase II detoxification enzymes, which markedly decline with age. LA appears to improve stress-response mechanisms by activating a transcription factor, Nrf2, enabling it to again bind to DNA sequences called the "Antioxidant Response Element" (ARE) found in over 200 genes involved in protecting cells against oxidative and toxicological insults. We are currently exploring why these stress-response mechanisms decline with age and are focusing on cellular signaling pathways that LA may induce to activate Nrf2-mediated gene expression.
We found that ALCAR and LA, when fed to old rats, markedly improve many indices of mitochondrial decay. Mitochondria may be the "Achilles heel" of cellular aging because their dysfunction adversely affects conversion of dietary fuels into useful energy, dysregulates cellular calcium levels, increases oxidative stress, and limits tissue renewal. Our goal is to determine whether these age-essential micronutrients can improve human health by maintaining mitochondrial function.
We are also interested in defining how LA and ALCAR improve such seemingly distinct aging lesions as mitochondrial decay and lost stress response mechanisms. We have evidence that these compounds synergistically regulate the metabolism of an enigmatic class of biomolecules called sphingolipids, which may be involved in both the age-related loss of Nrf2-mediated gene expression and mitochondrial decay. Identification that sphingolipids are part of these aging deficits opens the possibility for new therapies to improve human healthspan.
Since 2002, I have served as Director of the Environmental Health Sciences Center, which is funded by the National Institutes of Health to support research on the role of the environment in causing disease. The Center provides advanced technology that supports LPI researchers with the long-term goal of understanding how we can reduce our susceptibility to environmental stress as we age.
A major research project in my laboratory is aimed at understanding how oxidative stress, superoxide dismutase, and zinc are involved in Lou Gehrig's disease, also known as amyotrophic lateral sclerosis (ALS). ALS is a dreadful disease caused by the unexplained death of motor neurons that control the movement of all voluntary muscles. We have only about 500,000 motor neurons at birth that cannot be replaced. Mutations in the antioxidant enzyme superoxide dismutase are the first identified cause of ALS. Our research indicates that the loss of zinc from superoxide dismutase is what causes motor neurons to die. Using an animal model of ALS, we have found that a dietary deficiency in zinc can accelerate ALS development and that moderate supplements of zinc can extend survival. We are also investigating other dietary supplements, such as lipoic acid, acetyl-L-carnitine, and alpha-tocopherol, as possible means to slow the progression of ALS.
The second major project in the laboratory focuses on the roles of nitric oxide, peroxynitrite, and nitrotyrosine in human disease. The major function of superoxide dismutase is to scavenge superoxide, which is an oxygen radical. Nitric oxide also has a "dark side" and, following reaction with superoxide to produce the powerful oxidant peroxynitrite, can promote oxidative and nitrative damage to blood vessels, skin, heart, lung, kidney, and brain. We are characterizing the role of peroxynitrite in injuring cells and how cells respond to this damage. One sign of damage left by peroxynitrite is nitration of amino acids in proteins.
My lab examines genetic and epigenetic mechanisms of colorectal cancer development. We use human colon cancer cells and whole animal approaches, including transgenic models, to study mutational events in oncogenes and tumor suppressors (e.g., K-ras, beta-catenin, APC [Adenomatosis polyposis coli]), and the influence of dietary chemopreventive agents.
Our epigenetic work focuses on dietary phytochemicals as histone deacetylase (HDAC) inhibitors. Sulforaphane from broccoli, garlic organosulfur and organoselenium compounds, and a short-chain fatty acid derived from gut fermentation of dietary fiber (butyrate) inhibit HDAC activity in cancer cells and trigger growth arrest/apoptosis. We also observed HDAC inhibition and histone acetylation in peripheral blood mononuclear cells from healthy human volunteers who consumed a single serving of broccoli sprouts. We are now interested in translating this work into human subjects undergoing screening colonoscopies.
Cancer chemoprotection by dietary micronutrients, including vitamins and phytochemicals, is a very important component of our "war on cancer." Thirty to forty percent of cancers worldwide are preventable by optimizing diet, physical activity, and maintenance of appropriate body weight. Estimates are that cancer rates can be significantly reduced in lung (20-33%), stomach (66-75%), breast (33-50%), colon/rectum (66-75%), mouth/pharynx (33-50%), and liver (33-66%) by simple lifestyle choices, mostly related to diet, including adoption of a diet rich (400-800 g daily) in a variety of fruits and vegetables.
Few cancer prevention studies targeting the fetus or infant have been conducted, even though this early developmental stage is highly sensitive to cancer from chemicals crossing the placenta or transferred via mother's milk. Our laboratory focuses on the phytochemicals indole-3-carbinol (I3C) and sulforaphane, found in cruciferous vegetables (broccoli, cauliflower, Brussels sprouts). Initial studies utilized pregnant rodent models to ascertain potency, efficacy, and mechanism of action of these micronutrients, when added to the diet of the pregnant or lactating animal, in protecting her offspring from developing cancer later in life (even if they don’t eat their vegetables). We have repeatedly demonstrated that I3C in the maternal diet markedly protects her offspring from cancer from exposure in utero to environmental carcinogens. Ongoing studies will provide new insights into underlying mechanisms by which maternal fruit and vegetable intake protects against cancer in her offspring. We plan to move into translational studies with humans, correlating maternal diet, carcinogen exposure, and risk to the fetus or infant.
My research focuses on understanding the molecular mechanisms by which nutrient status affects the initiation and/or progression of chronic disease states like cancer. Low intake of several nutrients, such as zinc, could be a major risk factor for several types of cancer, as suggested by both epidemiological and laboratory studies. The main areas of interest in my laboratory are 1) the function of zinc across the lifespan and 2) dietary influences on prostate cancer development.
Zinc is a component of over 300 proteins, including DNA-binding proteins with zinc fingers, Cu/Zn superoxide dismutase, and several proteins involved in DNA repair, such as p53, which is mutated in half of human tumors. We have found that deficits in zinc intake could also have a major impact on an individual’s susceptibility to DNA damage and risk for developing cancer due to zinc’s function as an antioxidant and its role in DNA damage response. We are also interested in the effects of zinc during development and in the aging immune system.
Prostate cancer is one of the leading causes of cancer-related deaths in men. The prostate contain the highest concentration of zinc in the body, and low zinc intake may increase the risk for prostate cancer. We have also found that other dietary compounds, especially those found in traditional Asian diets, such as soy, teas, and cruciferous vegetables like broccoli, can limit prostate cancer development. A new, exciting interest in the laboratory is to understand the interaction between diet and epigenetic alterations in histone structure and prostate cancer risk.
The focus of my research is to use a "whole foods" approach to investigate the role of antioxidant and anti-inflammatory components of food in the prevention of chronic diseases, including diabetes and cancer. The specific aims of the research are to 1) elucidate the mechanisms by which antioxidant and anti-inflammatory nutrients reduce oxidative stress and prevent free radical-mediated diseases, 2) understand how nutritional deficiency in quantity and quality exacerbates oxidative stress and enhances the susceptibility to free radical-mediated diseases, and 3) understand the effect of a good diet in masking the expression of bad genes in the development of chronic diseases.
The type and quantity of dietary fat ingested affects human health, particularly the onset and progression of chronic diseases like diabetes, obesity, metabolic syndrome, and atherosclerosis. The liver plays a central role in whole body carbohydrate and lipid metabolism. Our research has focused on defining mechanisms for dietary fat regulation of hepatic carbohydrate and lipid metabolism. Our studies were the first to link dietary fat composition to the regulation of hepatic gene expression. These studies identified the peroxisome proliferator activated receptors-alpha, sterol regulatory element binding protein-1, carbohydrate regulatory element binding protein, and Max-like factor-X as targets of fatty acid regulation. These proteins are transcription factors that control the expression of multiple genes involved in carbohydrate and lipid metabolism. Dietary polyunsaturated fatty acid (PUFA) regulation of these transcription factors accounts, at least in part, for the well-known blood triglyceride-lowering (hypolipemic) effect of omega-3 fatty acids.
Our recent studies have focused on hepatic PUFA synthesis. We have used dietary fat manipulation and genetic approaches to identify a key enzyme involved in PUFA synthesis, i.e., fatty acid elongase-5 (Elovl5), as an important regulator of both hepatic and plasma fatty acid composition and triglyceride levels. Changes in hepatic Elovl5 expression were also found to control hepatic carbohydrate metabolism and blood glucose levels. These studies established that products of hepatic PUFA synthesis control both hepatic triglyceride and glucose production.
Future studies will determine whether changes in Elovl5 expression or the expression of other fatty acid elongases affect the onset of atherosclerosis or diabetes. These studies may identify novel approaches to control aberrant blood lipid and glucose levels associated with the progression of chronic metabolic diseases.
Our laboratory is interested in bioconjugation reactions involving lipid peroxidation (LPO) products and the relevance of these reactions to human health and disease. LPO products are formed when fats are oxidized (become "rancid") by reactive oxygen species. Some LPO products contain reactive moieties, notably 2-alkenals and epoxides, that can cause cellular damage by covalent reaction with proteins and DNA. The human body has several defense mechanisms in place to remove the reactive nature of these toxic LPO products: reduction of lipid hydroperoxides and aldehydes, and bioconjugation with glutathione. If these defense mechanisms are compromised, however, escaped LPO products may cause cellular damage to proteins, oxidative modification of low density lipoprotein (LDL), and alkylation of DNA, ultimately leading to cardiovascular disease and cancer.
We are particularly interested in the role that vitamin C (ascorbic acid) plays in LPO and how vitamin C can mitigate the damaging effects of LPO products. We have found that ascorbic acid promotes the detoxification of the LPO product, 4-hydroxy-2-nonenal (HNE), in cultured human monocytes—precursors of macrophages and atherosclerotic foam cells—by increasing conjugation of HNE with glutathione and increasing its cellular export. These vitamin C-induced effects resulted in a 30% decrease of damage to cellular proteins. In another study, we exposed cultured human monocytes to the cigarette smoke toxin, acrolein, and found that ascorbic acid itself forms a conjugate with acrolein. The ascorbic acid-acrolein conjugate was further metabolized into a stable deoxyketose. These findings indicate that vitamin C conjugation may contribute to the elimination of 2-alkenals in a cellular environment. Vitamin C reaction with reactive products is not uncommon in nature. According to a literature survey we conducted in 2009, there are at least two dozen examples of naturally occurring reaction products of ascorbic acid with plant metabolites. We will determine to what extent vitamin C conjugation contributes to the detoxification of oxidized lipids in humans and whether vitamin C and glutathione conjugates have diagnostic value as biomarkers of oxidative stress.
Our research is focused on understanding the importance of the regulation of antimicrobial peptide expression by the vitamin D pathway. The current model proposes that when immune cells like macrophages encounter a pathogen and become activated, the vitamin D pathway is turned on, leading to the induction of the cathelicidin antimicrobial peptide if serum levels of vitamin D are sufficient. We have shown that this mechanism is conserved in humans and primates but not in other mammals. Therefore, we developed a transgenic mouse that carries the human cathelicidin gene, which is properly expressed and responds to vitamin D. Using this model, we are testing the ability of vitamin D to protect against pathogens like Mycobacterium tuberculosis—the pathogen that causes tuberculosis—that infect macrophages. Vitamin D has been used to treat tuberculosis, and its deficiency is associated with increased risk of tuberculosis. This model will allow us to test the role of vitamin D and cathelicidin during initial infection, latency, and reactivation. We will investigate other infectious diseases as well.
Another focus of our research is to identify additional nutritional compounds and small molecules that regulate the expression of the cathelicidin gene. In addition to vitamin D, the cathelicidin gene is induced by sodium butyrate and lithocholic acid. Lithocholic acid functions through the vitamin D receptor. Nutrients that bind the vitamin D receptor may modulate the immune system by inducing the cathelicidin gene. A 50,000-small molecule library is being screened for regulators of the cathelicidin gene. The identification of new regulatory compounds may give clues as to how the gene is regulated in vivo and lead to the identification of other nutrients that can be used to boost the immune system.
Finally, we are interested in determining the impact that vitamin D insufficiency or deficiency has on the function of the innate immune system in the elderly, who are often vitamin D deficient. Aging is accompanied by a low-grade, chronic, systemic up-regulation of inflammation, and vitamin D has important anti-inflammatory properties. We hypothesize that this excess inflammation may be due, in part, to vitamin D deficiency. We are interested in determining if restoration of sufficient levels of vitamin D will reduce the inflammatory phenotype. We also want to determine if reversing severe deficiency will raise cathelicidin protein levels in the blood. Our research has shown that high levels of the cathelicidin protein in the blood of kidney dialysis patients and sepsis patients may be protective against poor outcomes from infection or sepsis. Both of these conditions predominantly afflict the elderly.
My laboratory focuses on identifying biomarkers in humans and parallel animal models that are associated with colorectal and pancreatic cancer and can be modified by diet and dietary supplements, including micronutrients and polyphenols. Colorectal cancer is an important public health problem, annually leading worldwide to over 500,000 deaths and in the U.S. nearly 150,000 new cases and 50,000 deaths. Dietary change, both feasible and safe, represents a viable strategy for preventing colorectal cancer; however, dietary intervention trials often showed no protection. There is a need for biomarkers of exposure, risk, and response to dietary interventions. Such biomarkers will provide crucial data to 1) identify individuals at increased risk or early stages of colorectal carcinogenesis, 2) measure compliance with dietary interventions, and 3) predict individuals most likely to benefit from a long-term dietary intervention (i.e., personalized cancer prevention).
In collaborations with researchers at the National Cancer Institute, Pennsylvania State University, Ohio State University, and Texas A&M University, we have been linking prospective NCI-funded human cohort and nutrition studies to USDA and Tufts food databases to identify diets and dietary components, most notably flavonols, with promise of efficacious cancer prevention. For dietary components showing efficacy, we have been identifying biomarkers of exposure, risk, and early dietary response, especially interleukin 6, through parallel human intervention and animal model studies in serum, feces, and tissue. The functional significance of the identified molecular targets in carcinogenesis will be tested using cell culture and transgenic mouse studies. The goal is to use the identified molecular targets and biomarkers in clinical trials to test the efficacy of diets and dietary supplements for cancer prevention.
The flavin-containing monooxygenases (FMOs) and cytochrome P450s (CYPs) are enzymes that oxygenate many of the drugs and chemicals that we ingest, inhale, or absorb. I am interested in the role these enzymes play in modulating disease and the impact of genetic variants on metabolism, especially in two cases: sulindac and FMO2.
Sulindac is an anti-inflammatory drug used to treat individuals with familial adenomatous polyposis (FAP), an inherited disorder characterized by colorectal cancer. FMO3 oxygenates sulindac sulfide, leading to its increased excretion. Studies have shown that among individuals with FAP, those with FMO3 polymorphisms that have a reduced capacity to oxygenate and excrete sulindac have a reduced polyp and tumor burden. A human study has demonstrated that dietary consumption of Brussels sprouts decreases conversion of trimethylamine to trimethylamine N-oxide, another reaction metabolized by FMO3. We are investigating the hypothesis that consumption of Brussels sprouts will reduce and delay sulindac oxygenation. If this is correct, dietary regulation of FMO3 could become a strategy for enhancing and prolonging the efficacy of sulindac.
Whereas FMO3 is the predominant FMO in human liver, FMO2 is the dominant isoform in lung. However, only some individuals of African or Hispanic/Latino descent produce active protein; all other populations have a polymorphism (genetic variation) for FMO2, rendering them with little capacity for metabolism of FMO substrates in the lung. Several classes of anti-tuberculosis drugs require activation by FMOs for bactericidal activity. Mycobacteria (causative agents for TB and leprosy) have their own FMO that activates these drugs. We have hypothesized that people with active FMO2 who have TB and are treated with these drugs will have a different drug response (toxicity and efficacy) than people without active FMO2. If our hypotheses are confirmed in mice with and without FMO2, physicians may then have a strategy to tailor drug therapy for TB on the basis of FMO2 polymorphism.
We investigate the lipid-regulating properties of natural dietary constituents and their therapeutic potential against chronic metabolic diseases, such as obesity and type 2 diabetes, and associated cardiovascular dysfunction.
Currently, our research focuses on hypertriglyceridemia (abnormally high blood triglyceride levels), a type of dyslipidemia and a strong predictor of coronary heart disease. Although epidemiologic and intervention studies clearly show that lowering blood triglycerides decreases cardiovascular events and mortality rates, over 65 million Americans have hypertriglyceridemia. This paradox points to major limitations in the effectiveness of current anti-hypertriglyceridemic therapies, which mainly rely on fibrate drugs when lifestyle changes have not been successful. However, side effects associated with fibrates remain a concern. Therefore, safer alternative therapies to mitigate hypertriglyceridemia are needed.
There is limited yet compelling evidence that lipoic acid, a natural compound in food, lowers triglycerides in humans, and could offer new solutions to this health problem. Our animal studies support this notion and show that lipoic acid prevents severe hypertriglyceridemia and decreases abdominal fat mass in otherwise obese rats. Further experimental evidence suggests that lipoic acid acts via a mechanism distinct from fibrates and is without fibrate-associated side effects. Mechanistically, lipoic acid improves blood triglycerides by the concerted inhibition of hepatic triglyceride synthesis and stimulation of triglyceride clearance in peripheral tissues. Through biochemical, cell culture, and animal studies, we hope to further uncover the molecular mechanism of action of lipoic acid, thereby allowing the rigorous testing of lipoic acid’s effectiveness vs. fibrate drugs in humans.
Drugs and environmental toxins, as well as some vitamins, herbs, and other bioactive food components, are metabolized and excreted from the body by a complex network of enzymes and transport proteins, referred to as xenobiotic pathways. One key focus of our research is to define the specific enzymes and transporters involved in the metabolism and excretion of certain vitamins and other bioactive food components. An additional focus in this area is determining the ability of vitamin E and other bioactive food components to modulate xenobiotic enzymes and transporters and possibly alter the metabolism and elimination of drugs and environmental or occupational toxins.
Polyaromatic hydrocarbons (PAHs) are environmental toxins that occur from both man-made and natural sources. High-level occupational exposure to PAHs has been shown to increase cancer incidence in exposed workers. Our recent data suggest that high-dose vitamin E supplementation may provide protection from PAH exposure by modulating PAH metabolism and increasing excretion from the body. Defining the mechanism by which vitamin E provides protection from PAH exposure will not only increase our ability to protect PAH-exposed workers but also provide future research directions for determining the possible use of vitamin E for improving human health in non-occupational exposure situations, including in utero exposure.
A second research focus is determining the mechanism by which chemotherapeutic drugs, particularly platinum-based agents, cause neuropathy and the ability of antioxidants like vitamin E to prevent these neuropathies. Platinum-based chemotherapeutics are highly effective anti-cancer agents. However, platinum-induced neuropathies often force patients and their families to choose between quality of life vs. life span. Thus, determining the underlying mechanism by which these neuropathies develop will enable us to better design adjuvant therapies to prevent them.
Compelling evidence links oxidative stress and inflammation to major cardiovascular diseases (CVD), including atherosclerosis, hypertension, coronary heart disease, and stroke. While antioxidants and anti-inflammatories have been found to inhibit atherosclerosis in experimental animals, antioxidant vitamin trials to prevent or slow CVD in humans have been disappointing. The disparity between clinical trials and animal studies points to major gaps in our understanding of the effects and mechanisms of antioxidants in atherosclerotic vascular diseases in humans.
Our long-term goal is to target oxidative stress and inflammation in humans to better understand their etiologic roles in atherosclerosis. The intracellular redox environment regulates endothelial cell function; an imbalance in this redox environment may cause upregulation of cellular adhesion molecules and pro-inflammatory and pro-thrombotic mediators, all of which promote atherosclerosis. We have found that lipoic acid affects redox-sensitive cell-signaling processes, transcription factors, and gene expression in endothelial and mononuclear cells, and exerts strong anti-inflammatory and anti-atherogenic effects in experimental animals. In collaboration with researchers at Oregon Health & Science University, we are currently testing whether lipoic acid supplementation reduces risk factors for the development of atherosclerosis in humans by attenuating oxidative stress and inflammation.
