Research Highlight: Understanding Individual Differences in Chemical and Drug Metabolism

Environmental exposures affect health. 
Exposure science is the study of chemical, physical, or biological agents that humans come in contact with in their environments. Exposure information is needed to understand health risks from chemicals and other agents.

Evaluating the amount of specific chemicals people are exposed to allows scientists to estimate the dose of the chemicals people are getting. Knowing this allows researchers to develop studies to test whether that dose may affect human health. Results from exposure science studies are critical for developing interventions to prevent or minimize health hazards.

As our Center theme is gene-environment interactions, measuring exposures and understanding the dose is needed to be able to estimate the interaction of genes with chemical exposures.

Exposure science is known for its interdisciplinary approach. The Exposure Sciences ARE draws on the expertise of Exposure Sciences Program faculty within our department, Environmental and Occupational Health Sciences, including two faculty whose main appointments are in Environmental Engineering and Biostatistics. Two more Exposure Sciences ARE faculty come from the School of Pharmacy.

The Exposure Sciences ARE members work collaboratively with colleagues in the other CEEH cores to provide state-of-the-art methods in the use of various exposure assessment tools including biomarkers, a molecule in blood or urine that indicates a physiological or a particular disease state.

Exposure Sciences ARE member Yvonne Lin, a faculty in the School of Pharmacy, led a study this year to search for a biomarker in urine that would reflect the activity of a metabolic enzyme called cytochrome P450 2D6 (CYP2D6). This enzyme plays a role in metabolizing 15% of clinical drugs, including antipsychotics, antidepressants, beta-blockers, opioid analgesics, and dextromethorphan, a cough suppressant found in many over-the-counter cough and cold medicines.

CYP2D6 has wide genetic diversity. Some healthy people have no CYP2D6 activity (these folks are called Poor Metabolizers), while others have some activity and still others have extensive activity.
Phenotyping to test a person’s level of CYP2D6 activity is done with probe drugs or drug cocktails and blood draws. A safer, less invasive method would be to identify a biomarker, for example in urine, to measure the real-time activity of the CYP2D6 enzyme. This is what Lin and her team members did.

Child participants were given cough syrup.
One hundred healthy children were given the drug dextromethorphan, a common over-the-counter medication given to adults and children. Urine samples were collected before and after participants were given the drug. Samples were analyzed to try to identify urinary metabolites, small molecule fragments of the drug that could predict the child’s CYP2D6 phenotype.

Lin and her team were able to identify a candidate biomarker molecule that indicated the metabolism of dextromethorphan. The molecule was nearly absent in the 9 poor metabolizers and present in 91 participants in proportion to their CYP2D6 phenotype.

The team searched established databases to identify the molecule, but found no matches. They identified the biomarker by its mass, 443.3026 Da, and refer to it as Unknown 1, or Unk1. A paper about the study has been submitted and is currently in review.

Next steps are to identify Unk1 and validate it as a biomarker that can be used to measure clinical drug metabolism. This discovery has the potential to improve personalized medicine in children by providing a means to measure in a urine sample an individual ‘s response to the clinical drugs metabolized by CYP2D6.

Dr. Lin’s research interests include pharmacokinetics, the process by which a drug is absorbed, distributed, metabolized, and eliminated by the body; pharmacogenetics, genetic differences in metabolism that can affect individual responses to drugs; the regulation of drug metabolizing enzymes such as CYP2D6 in children; the effect of obesity and diabetes on the metabolism and elimination of clinical drugs, and metabolomics, the study of the chemical fingerprints and metabolites left behind in metabolism, as a tool to identify biomarkers.

Research Highlight: Studying the Links between Chemical Exposures, Genetics, and Neurodegenerative Disease

Lucio Costa, NND ARE leader
As the US population ages, more and more Americans suffer from neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis (ALS).1

Alzheimer’s disease is the 6th leading cause of death in the US. More than 5 million Americans are living with Alzheimer’s.2 In addition, over 500,000 Americans live with Parkinson’s.3

Approximately 5,600 Americans are diagnosed with ALS each year. The life expectancy of an ALS patient averages two to five years from diagnosis, and as many as 30,000 Americans have ALS at any given time. The National Institute of Neurological Disorders and Stroke (NINDS) reports that only 5–10% of ALS cases can be traced to genetics.4

Why and how these neurodegenerative diseases develop is for the most part unknown, and environmental factors such as the potential harmful effects of chemicals may play a role.
Investigators in the CEEH Neurotoxicology and Neurodegenerative Disease (NND) Area of Research Emphasis (ARE) study how certain chemicals can affect growth and development and how this might contribute to neurodegenerative diseases. They also look for genetic variations that might explain why some people are more susceptible to the harmful effects of chemicals than others, and work to understand the actual cellular, biochemical and molecular mechanisms of these neurotoxic chemicals.

Core leader Lucio Costa and Core member Clement Furlong study the gene family Paroxonase (PON), which includes PON1, PON2 and PON3. These genes provide the code that allows the body to produce enzymes to help break down certain kinds of pesticides in the body.

Recently Drs. Costa and Furlong investigated the level of one of these enzymes (confusingly named PON2 after the gene that codes for it) in mouse brain cells after the cells were treated with quercetin, a chemical compound found in fruits and vegetables. Previous research has shown that quercetin is an antioxidant that helps protect cells from the harmful effects of oxidative stress. Under normal conditions, the human body uses oxygen to fuel itself by combining the oxygen we breathe with the food we eat and digest. This process produces dangerous by-products such as “free radicals,” unstable atoms or molecules that wreak havoc in cells. Oxidative stress is a disturbance in the balance of free radicals and anti-oxidant defenses such as quercetin. Oxidative stress is associated with neurodegenerative diseases and also plays a role in age-related degeneration.5

Costa and Furlong’s team treated mouse brain cells (astrocytes) with quercetin. They found that these treated cells had higher levels of the PON2 enzyme, and that the PON2 levels increased in proportion to the concentration of quercetin. The team also measured the concentration of free radicals in the treated cells and found decreased levels of these. These findings suggest that quercetin increased the ability of the cells to destroy harmful free radicals and thus protect them from some of the damage caused by oxidative stress.

In cells taken from control mice that didn’t have the PON2 gene, and therefore didn’t produce the PON2 enzyme, quercetin was significantly less protective against oxidative stress. This suggests that the PON 2 enzyme plays an important role in helping quercetin protect us from free radicals. A next step in this research is to move from cells to animal models and study the effect of quercertin on PON2 levels in complex organisms.

Dr. Costa earned a degree in Pharmacology at the University of Milan and came to the US as a postdoctoral fellow at the University of Texas at Houston. As a post-doc, he was asked to discover why animals could become tolerant to the neurotoxic effects of organophosphorus insecticides. This research project sparked his lifelong interest in the myriad ways that chemicals can harm the nervous system.


Newsflash: Dietary Study Has Unexpected Results

© 2013, JupiterImages

Center member Dr. Sheela Sathyanarayana and her colleagues have published the results of an intervention trial that attempted to reduce exposure to endocrine-disrupting phthalates and Bisphenol A (BPA) in the diet. The article was released today in the Journal of Exposure Science and Environmental Epidemiology.

Phthalates and BPA are found in a wide variety of plastic products and contaminants in food. They can harm pregnant women and young children. The researchers wanted to find out if asking families to follow written instructions would lead to reduced exposure to these chemicals, compared to providing a catered diet specially prepared without plastics.

The study took place in summer, 2011 in Seattle. Eligible participants were 2-parent families with at least two 4-8 year-old children. Ten families with a total of 40 individuals were enrolled. The families were randomly assigned, half to the dietary replacement group and half to the written materials  group. The study lasted for 16 days.

Days 1-5 were Baseline Days during which the study was explained, instructions and materials such as glass food storage contains were provided, and detailed dietary questionnaires were completed for each family member every day. The written materials group was given written guidelines to reduce phthalate and BPA exposure, including descriptions of phthalates and BPA, sources of exposure with a focus on plastics, and suggestions for how to reduce exposure in daily activities. Urine samples were collected from all family members on Day 5.

Days 6-10 were Intervention Days. The dietary replacement group was provided with catered food from a local caterer who used fresh, local and, whenever possible, organic ingredients. The food was prepared, stored and transported without using plastics. Both groups were instructed to use filtered water, consume beverages from non-plastic containers when possible, use non-plastic utensils and dishware, and store foods in the glass storage containers provided. Dietary surveys were completed every day. A urine sample was collected for each family member on Days 9 and 10.

Days 11-16 were Post-Intervention Days. Dietary surveys were completed every day and urine was collected on Day 16.

Here's what they found:  There was a significant increase in phthalate metabolites during the intervention period compared to the baseline period for the group that ate a catered diet. The average phthalate level in the dietary replacement group when they ate the catered diet (Days 9 and 10) was 25 times the level at baseline (Day 5). This was a complete surprise! In contract, they found no change in urinary phthalate metabolites in the written materials intervention group between baseline and intervention.

Since the increase in urinary phthalate concentration in the dietary replacement group was unexpected, the researchers tested the food ingredients in the dietary replacement group to see if there was phthalate contamination. They found that the dairy products milk and cream had phthalate concentrates above 440 ng/g, and that the spice mix had very high concentrations, 700 ng/g in ground cinnamon and cayenne pepper and the astounding level of 21,400 ng/g in ground coriander. All other ingredients had phthalate concentrations in the range reported in the literature.

The researchers had hoped to see reductions in urinary phthalate metabolites in both groups, but expected that providing written materials would be less effective than providing catered organic food prepared without exposure to plastics. But that was not what they found. Instead, they concluded that accepted methods to reduce dietary exposure to phthalates and BPA (e.g. minimize contact with plastics) may not actually reduce urinary concentrations of these chemicals. This study highlights how contaminated foods can contribute to excessive phthalate exposure. However, it is not known whether this was a isolated and rare contamination event or whether the food supply is systematically contaminated with high phthalate concentrations.

With no change in phthalate and BPA levels between baseline and intervention days in the group who received written materials, the trial supported the team's hypothesis that providing written recommendations to reduce dietary exposure to plastics is insufficient. This is corroborated in primary practice where it has been shown that written guidelines are ineffective to change health-related behaviors.

Families can focus on buying fresh, low-fat foods and avoiding plastic packaging and dishware. But this study demonstrates that it may take federal regulation to eliminate phthalates from the food supply.