The research in our laboratory focuses on understanding the pathogenesis of atherosclerosis (hardening and thickening of arterial walls leading to heart attacks and strokes) and the modulatory effects of antioxidants, essential minerals, and other micronutrients. One of the earliest events in atherosclerosis is dysfunction of the endothelium (a single cell layer lining arterial walls). An important consequence of endothelial dysfunction is recruitment of white blood cells called monocytes to the arterial wall, where they initiate chronic inflammation. In addition, endothelial dysfunction leads to impaired vasodilation (blood vessel relaxation), which is a causal factor in hypertension. We are performing biochemical, cell biological, animal, and human studies to investigate the mechanisms and consequences of endothelial dysfunction, the role of pro-oxidant transition metal ions, such as iron and copper, in this process, and the effectiveness of antioxidants and anti-inflammatories in ameliorating endothelial dysfunction, arterial inflammation, and atherosclerosis.
In collaboration with investigators at Boston University, we found that vitamin C improves endothelial function and vasodilation in patients with heart disease or coronary risk factors and lowers blood pressure in moderately hypertensive patients. Work in our laboratory using human aortic endothelial cells showed that metal chelators (metal-binding agents) used in "chelation therapy," lipoic acid, and flavonoids, but not vitamin C, inhibit the expression of adhesion molecules, which are responsible for monocyte recruitment to the arterial wall in atherosclerosis. We also found that metal chelators and lipoic acid inhibit adhesion molecule expression, inflammation, and atherosclerosis in transgenic mouse models and are now investigating the effects of lipoic acid supplementation in humans. The ultimate goal is to find new, effective strategies to prevent and treat heart disease, stroke, and chronic inflammation by diet and lifestyle modifications and dietary supplements.
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 a specific biochemical pathway or enzyme does not require 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 to us. 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 an alpha-TTP-knockout mouse in which the gene for this protein has been deleted. Hence, this mouse is unable to maintain normal alpha-tocopherol concentrations, especially in the brain, and becomes vitamin E deficient. Plans are under way to use this mouse to define the role of alpha-tocopherol in protecting the brain from neurodegenerative diseases.
Studies of vitamin E metabolism serve to determine how the body responds to excess vitamin E. We are currently investigating how the liver protects the body against accumulating too much vitamin E and how these mechanisms might have adverse effects with respect to pharmaceutical drug metabolism. Overall, the assessment of the delivery and function of vitamin E in humans has lagged because previously we lacked the appropriate tools. The availability of deuterated tocopherols has now made it possible to carry out measurements of the biokinetics and bioavailability of vitamin E in humans.
Our laboratory has developed new methodologies using liquid chromatography-mass spectrometry that are 100-fold more sensitive than previous methods. The purpose of these measurements is to detail the requirements of normal humans and those with diseases related to oxidative stress, such as obesity, atherosclerosis, cancer, diabetes, and Alzheimer's disease.
Our research seeks to identify the mode of action of two "age-essential" micronutrients, lipoic acid (LA) and acetyl-L-carnitine (ALCAR), which appear to maintain vitality into advanced old age. 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. As the aging process is highly complex and only limited knowledge now exists regarding its underlying mechanisms, we are also using LA and ALCAR as nutritive "keys" to unlock important mechanisms associated with the basic biology of aging. It is our hope that better understanding of these fundamental events may also lead to effective therapies for a number of age-related diseases and provide a better quality of life.
We found that ALCAR and LA improve two of the most important cellular lesions of aging, namely the inability to respond to oxidative and toxicological challenges, and also the loss of mitochondrial function. Of the former, feeding old rats LA markedly elevates both cellular ascorbic acid and glutathione levels, and also induces "Phase II" detoxification enzymes, which otherwise markedly decline with age. This LA-induced improvement in our flagging cellular defense machinery significantly enhances the ability to respond to many types of challenges. 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 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.
In addition, we found that ALCAR and LA, when fed to old rats, markedly improve many indices of mitochondrial decay. This is important, as mitochondria may be the "Achilles' heel" of cellular aging because their dysfunction would adversely affect conversion of dietary fuels into useful energy, dysregulate cellular calcium levels, increase oxidative stress, and limit tissue renewal. It is now our goal to determine whether these age-essential micronutrients can also 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 now 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 anti-aging therapies to improve human healthspan.
For the past three years, 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 all 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, and they 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 and acetylcarnitine, 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.
This laboratory investigates diet and lifestyle factors that affect the development of colorectal cancer, and the importance of both genetic and epigenetic events. In the genetic area, we are studying the molecular pathways that affect beta-catenin/Tcf signaling, which is commonly dys-regulated at an early stage in human colon cancers. We use 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and related cooked meat-derived mutagens to examine beta-catenin mutational changes in colon (and other) tumors, coupled with knockout/transgenic models and human colon cancer cell lines.
Superimposed on these studies, we examine mechanisms of cancer prevention/therapy by dietary phytochemicals. Tea polyphenols, chlorophylls in green leafy vegetables, and indoles in cruciferous vegetables have been found to exhibit anticancer properties, in part through inhibitory effects on beta-catenin signaling, and these dietary agents are now being included in human trials using ultra-low dose exposure protocols. In the area of epigenetics, we are studying sulforaphane from broccoli and allyl compounds in garlic as inhibitors of histone deacetylase (HDAC) activity. HDAC inhibitors are of growing interest as both cancer chemopreventive and therapeutic agents, acting on the "histone code" to re-express epigenetically silenced genes such as P21 and bax, thereby triggering cancer cells to commit suicide (via apoptosis). We collaborate with the Ho laboratory on translational studies in humans, showing HDAC inhibition in circulating blood cells of healthy human volunteers who consumed sulforaphane-rich broccoli sprouts.
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. Epidemiologically, populations from economically developing countries exhibit higher rates of cancer of the stomach, liver, cervix, and upper GI tract, whereas rates of cancers of the breast, prostate, colon, and rectum are higher in developed countries. The World Cancer Research Fund and American Institute for Cancer Research suggest that cancer rates can be significantly reduced in the 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.
Most cancer studies have targeted adults. Few, if any, cancer prevention studies have been conducted in utero for the infant and young child, even though many studies have documented that this early developmental stage is highly sensitive to the carcinogenic effects of chemicals capable of crossing the placenta or transferred through mother's milk. Our laboratory addresses this important area of research, initially focusing on three classes of phytochemicals that have been extensively studied in the LPI: 1) indoles from cruciferous vegetables (broccoli, cauliflower, Brussels sprouts), 2) polyphenols from green tea, and 3) chlorophylls, the natural green plant pigments found in green and leafy vegetables. Initial studies have focused on pregnant rodent models to ascertain potency, efficacy, and mechanism of action of these phytochemicals in protecting the fetus from transplacental carcinogenesis of the liver and lung by chemicals found in the environment. These studies will provide new insights into the underlying mechanisms by which fruit and vegetable intake and specific phytochemicals reduce cancer risk in humans. Our preliminary results suggest that mice born to mothers consuming indole- 3-carbinol from cruciferous vegetables during pregnancy and lactation are significantly protected from carcinogenicity.
The primary interest in our laboratory lies in understanding the mechanisms through which certain plant-based chemicals can inhibit cancer in experimental animals and the extrapolation of those findings to human cancer intervention. Current emphasis is on the study of chemoprevention by natural chlorophylls and their watersoluble chlorin derivatives. A hallmark for this laboratory has been its development and use of a lower vertebrate animal model, the rainbow trout, which has certain attributes allowing unique contributions to this field of research. Recent studies have applied dose-dose matrix designs with up to 10,000 individuals to quantify the complex interrelationships between carcinogen dose applied, anti-carcinogen dose applied, amount of DNA damage in the target organ, and final tumor outcome in that organ nine months later. These approaches have provided the most elaborate database available in any animal studies on the utility of carcinogen-DNA adducts as early biomarkers for the prediction of anti-carcinogen effects on eventual cancer incidence. For instance, we recently demonstrated the conditions under which reduction in aflatoxin-DNA damage by dietary chlorophyllin could precisely predict eventual reduction of aflatoxin-driven liver cancer in the trout model. These findings were then translated into a human intervention trial in rural China, where aflatoxin exposure is a major risk factor for liver cancer and one in ten adults dies of this disease. This trial demonstrated that dietary chlorophyllin reduced urinary aflatoxin-DNA adduct biomarkers in humans as effectively as it did in the trout and has pointed to an affordable, low-risk approach for reducing risk of this dreaded disease in China, Southeast Asia, and Africa.
Our current chemoprevention research focuses on the following set of questions: a) what exactly are the mechanisms for cancer prevention by chlorophyllin and its individual chlorin components; b) how will these mechanisms apply to other animal models (rats, mice), to other cancers (lung, stomach, mammary) besides liver, and to humans; and c) will natural chlorophylls found in our foods provide a degree of protection comparable to chlorophyllin? These studies are being carried out in a program project in close collaboration with two other LPI investigators, Drs. Dashwood and Williams. Encouraging results in the past year indicate that natural chlorophyll, at reasonable dietary levels, can strongly inhibit cancer initiation in trout and rats, through a simple mechanism currently being tested in human volunteers. We hope that the results of this research will identify phytochemicals effective at reducing risks for lung, mammary, and stomach cancer induced by polyaromatic hydrocarbon exposure, colon cancer induced by heterocyclic amines, and aflatoxin-related liver cancer in humans.
My research focuses on understanding the molecular mechanisms by which nutrient status affects the initiation and/or progression of chronic disease states, such as 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 the laboratory are:
Zinc, oxidative stress, and DNA integrity: 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. It can be hypothesized that insufficient zinc intake will impair antioxidant defenses and compromise DNA repair mechanisms, making the cell highly susceptible to oxidative DNA damage. Thus, deficits in zinc intake could also have a major impact on an individual’s stress response and risk for developing cancer. We are identifying molecular mechanisms by which zinc acts as an antioxidant and protects us from environmental challenges.
Dietary influences on prostate cancer development: A significant portion of the population is at risk for marginal zinc deficiency and potential risk for cancer. The prostate contains the highest concentration of zinc in the body and is sensitive to zinc fluctuations. However, the function of zinc in the prostate remains unknown. Low zinc intake could dramatically increase one’s risk for developing prostate cancer. Zinc acts to combat oxidative damage, plays a role in DNA repair, and also bears important anti-inflammatory and anti-proliferative properties. The ability of zinc to target multiple points of the carcinogenic pathway and its unique importance in the prostate could make zinc an effective chemoprevention agent. We are also interested in other dietary compounds, such as soy, teas, and cruciferous vegetables, that have antioxidant, antiinflammatory, and anti-proliferative properties on limiting prostate cancer development. An exciting new interest in the laboratory is understanding the interaction between diet, epigenetic alterations in histone structure, and prostate cancer risk.
Identification of zinc status biomarkers: Currently a reliable marker for zinc status does not exist. Using genomic and molecular approaches, we are probing for novel markers for zinc deficiency in humans.
There are three main areas of research in our laboratory:
Antioxidants and free radical metabolism in health and disease: The focus of this research is to use a "whole food" approach in investigating the role of antioxidants and anti-inflammatory properties of food in the prevention of chronic diseases, including diabetes and cancer. The specific aims of the research are: 1) to elucidate the mechanisms by which antioxidant and anti-inflammatory nutrients reduce oxidative stress and prevent free radical-induced diseases, and 2) to understand how nutritional deficiency in quantity and quality exacerbates oxidative stress and enhances the susceptibility to free radical mediated diseases.
Nutrition and gene expression: The focus of this area of research is to investigate the interaction of nutritional status and genetic expression in disease prevention and health promotion. The specific aims are to understand the importance of nutrients in controlling the gene expression of transcription factors, NFkkappaB and AP-1, which influences the immune defense system, and to study the interaction of nutrition and the expression of the first-line antioxidant enzyme, CuZnSOD, in normal and disease states.
Food-borne toxins in the pathogenesis of lung disease: The goal of this research is to understand the role of nutrition in the pathogenesis of acute pulmonary edema and emphysema. We focus on the interaction of antioxidant nutrients and microbial ecology, mechanism of action, sequence of toxicological events, and tissue selectivity of toxicity and prevention strategies for lung disease.
The type and quantity of dietary fat ingested have multiple impacts on human health, particularly the onset and progression of chronic diseases. Low-fat, high-carbohydrate diets promote the synthesis of saturated and monounsaturated fats by the liver. Diets enriched in saturated fat promote the onset of atherosclerosis, obesity, and diabetes. Diets deficient in n-3 and n-6 polyunsaturated fatty acids affect blood triglycerides, inflammation, reproduction, learning, and vision.
The liver plays a central role in whole body carbohydrate, lipid, and cholesterol metabolism. Metabolic pathways that convert carbohydrate to fatty acids or cholesterol or modify fatty acid structure likely contribute to changes in blood lipids associated with chronic disease. For many years, our research focused on the role exogenous (dietary) fat played in modifying the expression of genes involved in carbohydrate and lipid metabolism. Our studies were the first to link dietary fat composition to the regulation of hepatic gene expression. These studies revealed three operative mechanisms in the liver that were controlled by dietary fatty acid composition: peroxisome proliferator activated receptors- a, sterol regulatory element binding protein-1, and a heterodimer composed of carbohydrate regulatory element binding protein and Max-like factor-X. Dietary fat controlled the activity or nuclear abundance of these transcription factors, which in turn controlled the expression of multiple genes involved in carbohydrate and lipid metabolism. In this fashion, dietary fat functioned as a feedback and feed-forward regulator of specific metabolic pathways that affect hepatic and whole body lipid composition.
More recently, we have turned our attention to key metabolic pathways that alter the type of fatty acid synthesized in the liver. These pathways include de novo lipogenesis, fatty acid desaturation, and fatty acid elongation. We have recently established that the expression and activity of fatty acid desaturases and elongases are modified in vivo in mouse models of diabetes, obesity, and fatty liver. Using a genetic approach, we altered the expression of specific fatty acid elongases or desaturases in primary rat hepatocytes or in mouse liver. Studies in mice have revealed that changes in hepatic expression of these enzymes alter both hepatic and blood lipid composition. Future studies will determine whether changes in expression of specific fatty acid elongases or desaturases have any impact on the onset of chronic diseases like atherosclerosis and diabetes. The outcome of these studies may provide novel strategies to control blood lipids and their impact on chronic diseases.
Our laboratory is interested in bioconjugation reactions involving lipid peroxidation (LPO) products and in 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.
Vitamin C (ascorbic acid) is well known as an antioxidant. Other researchers have proposed that ascorbic acid can promote the formation of LPO products by reducing lipid hydroperoxides to form reactive intermediates that decompose spontaneously into 2-alkenals. After confirming this type of interaction between ascorbic acid and lipid hydroperoxides, we discovered that ascorbic acid has the ability to form conjugates with 2-alkenals in test-tube experiments. In addition, mass spectrometric measurements provided evidence supporting the presence of vitamin C conjugates with the LPO-derived 2-alkenals, 4-hydroxy-2- nonenal (HNE) and 2-propenal (acrolein, ACR), in human plasma and urine. Interestingly, humans and chimpanzees contain relatively high levels of the ascorbic acid-HNE conjugate (AscHNE), whereas all other animals tested so far show low or undetectable levels of AscHNE. A present research focus is to determine why there is a clear species-dependent difference in AscHNE plasma levels.
In collaboration with LPI researcher Dr. Maret Traber, we have found that the average plasma level of AscHNE is about 60% lower in smokers compared to non-smoking age-matched control subjects. Moreover, AscHNE levels increased more than ten-fold after extreme physical activity, as seen with ultramarathon runners after a 50 km-race. These data raise the suggestion that AscHNE can be used as a biomarker of the physiological response to oxidative stress. This idea will be further tested in other in vivo models of oxidative stress. Finally, our group has initiated studies to determine the biological effects of LPO products and their bioconjugates on vascular function. The rationale for these studies with cultured endothelial cells is that LPO products are known to be involved in the early steps leading to atherosclerosis.
