Concentration of total mercury and methylmercury in commercial cigarettes: Examining routes of human exposure.

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1 Concentration of total mercury and methylmercury in commercial cigarettes: Examining routes of human exposure. Forrest Wilder Campnell Mentors: Todd O hara and John Harley Undergraduate of Biochemistry, UAF Winter-Spring Formatted for submission to URSA 1

2 Abstract: Exposure to mercury (Hg) during development and maturation periods can be compromising to the central nervous system of young children in Mexico (Acosta-Saavedra et al., 2011). In this population, total Hg concentrations ([THg]) in pregnant women were found to increase with both tobacco exposure and seafood consumption (Gaxiola-Robles et al., 2014). Therefore, understanding sources of Hg exposure are important for the wellbeing of foreign and domestic populations. The overarching goal of this ongoing investigative project is to determine the sources of Hg exposure to the La Paz Mexican population. If cigarettes are significant covariates in high [THg] hair samples, then they will have detectable Hg concentrations and [MeHg]. To investigate this hypothesis, a Direct Mercury Analyzer (DMA-80) and a MERX-M will be used to analyze cigarette and hair samples of interest in the Wildlife Toxicology Lab. If time allows, more ideas will be investigated such as continued studies in the absorption of Hg present in tobacco smoke onto hair samples treated with and without detergent. 2

3 Goals: To investigate the hypothesis of my ongoing undergraduate research, several specific aims have been identified: Aim 1. Continue to quantify [THg] in homogenized cigarettes purchased in La Paz, Mexico and Fairbanks, Alaska using a Direct Mercury Analyzer (DMA-80) Aim 2. Continue to assess if detected [THg] in cigarettes are at a level to suggest it is a significant covariate in the proposed models of Gaxiola-Robles et al. (2014). There is scarce literature detailing concentrations of Hg found in commercial cigarettes. However, we can compare the [THg] in cigarettes within the forty year time period since the 1976 Suzuki study as well as examine additional options to study sources of Hg exposure in conjunction with data from our current collaborators in La Paz, Mexico. This includes Hg absorption into hair samples. Aim 3. Quantify [MeHg] in cigarettes purchased in La Paz, Mexico and Fairbanks, Alaska using a MERX-M Little information is known about the most lethal form of Hg (Clarkson & Magos, 2006) in commercial cigarettes besides the 1976 Suzuki study. Using the newly acquired MERX-M in the Wildlife Toxicology Lab would be an excellent source of information to gauge Hg toxicology in cigarettes. Background and Significance: The toxicity of mercury (Hg) on some model organisms are well established in scientific literature; however, finding sources of human exposure is a large concern for federal organizations such as the Federal Department of Agriculture (FDA) and the Environmental Protection Agency (EPA). These concerns stretch across national borders to 3

4 countries where Hg concentrations in human and wildlife may exceed established domestic thresholds. Studying sources of Hg exposure can guide governmental organizations in mitigating unwanted sources of Hg from negatively affecting biological systems. Cigarettes are composed of a cocktail consisting of carcinogenic and toxic chemicals, including Hg (Suzuki, 1976). However, literature examining for the total concentrations of Hg in even the most commonly bought cigarettes is sparse, making it difficult to assess if cigarettes are sources of high Hg concentrations measured in domestic or foreign populations of interest. A recent study on pregnant women in Baja, Mexico revealed that 72% (n=75) exceeded the 1 µgg -1 recommended EPA Hg concentration in hair (Gaxiola-Robles et al., 2014). Although the higher concentration is partially attributed to a high seafood diet, 32% answered that they were exposed to tobacco smoke regularly (Gaxiola-Robles et al., 2014). Additionally, Gaxiola-Robles et al. (2014) reported that 43% of the variation seen in [THg] could be explained by including tobacco exposure in the model in conjunction to seafood consumption and BMI. This highlights the need for additional research of tobacco exposure in relation to [THg] found in the hair samples. To understand the routes of exposure of Hg to account for the high concentration in La Paz women, the total concentration of Hg in locally bought cigarettes should be examined. It is imperative to know what chemical forms and compounds would be relevant to the topic. A review on the toxicology of Hg compounds examines the different toxicologies of Hg in its elemental and organic state (Clarkson & Magos, 2006). Methylmercury (MeHg) is the most toxic to humans, with seafood being a high contributor to human exposure (Clarkson & Magos, 2006). While the inorganic states (Hg 2+, Hg 0 ) are not as potent, they are still harmful to human health. Methylmercury targets the central nervous system, and concentrations that were found in Gaxiola-Robles et al. (2014) have been associated with negative health outcomes in other populations (i.e.6 µgg -1 increase in hair THg correlated with increasing 4

5 levels of tremors in Greenlandic Eskimo populations; Auger et al., 2005). Elemental Hg vapor, such as that potentially volatilized by the burning of a cigarette, is infamous for causing psychological problems such as Mad Hatter s disease (O Carroll et al., 1995). This further establishes the importance of understanding the Hg composition in cigarettes. One study found that a single cigarette could release 5-7 ng of Hg in smoke, the percentage at which exists as MeHg is stated to by unknown (Suzuki, 1976). The only source of methylmercury in cigarettes was found in the filter which was disregarded as the researchers concluded it would not affect Hg concentrations because it does not enter the body (Suzuki, 1976). Furthermore µg -1 of Hg was found in Japanese cigarette brands while µg -1 was found in foreign brands (Suzuki, 1976). During summer of 2015, I found that there was µg/kg of THg in La Paz cigarettes with insignificant differences between the brands themselves. These values are much lower when compared to the Suzuki et al. (1976) THg values of cigarettes ( µg/kg), but I feel it is worthwhile to compare these results to cigarettes purchased here in Fairbanks. As indicated by Gaxiola-Robles (2014), there is an unaddressed correlation between the interaction of tobacco and seafood exposure to total Hg concentration found in hair samples of Mexican individuals. This study would be the first step in determining if the observed correlation is consistent and can be explained by [THg] found in cigarettes. The only study to address [THg] in cigarettes was performed in 1976, possibly being outdated since tobacco farming and production methods have been the main cause of suspicion of Hg contamination in cigarettes (Suzuki, 1976). Depending on the results, it may also serve as a precursor to shed some light and expand scientific knowledge on the dangers of second hand smoke and the use of cigarettes in general, as well as currently accepted levels of [THg] by federal regulators 5

6 Design and Methodology: In order to meet my specific aims for this study, tobacco from cigarettes purchased in La Paz, Mexico and Fairbanks, Alaska will be homogenized using a Cryomill grinder that embrittles and preserves volatile chemicals. Total mercury in the homogenized samples will be measured using a direct mercury analyzer in the O Hara lab (DMA-80, Milestone, Shelton, Connecticut), using methods similar to those used to measure Hg in hair segments in Gaxiola-Robles (2014). At the beginning of each run, blanks and aqueous standards will be analyzed for quality assurance and control. Multiple cigarette brands were purchased locally in La Paz, Mexico with multiple individual cigarettes, allowing for sufficient samples for multiple replicates to ensure precise measurements. The mean value for all replicates will be used in the statistical analysis, and I will subsequently use a Tukey honest significance test that compares the means of every treatment to the means of every other treatment. For my ongoing research with Hg absorption into hair samples itself, dog hair obtained advantageously through the Vet Med department will be measured with the DMA-80 to establish a blank [THg] measurement before exposure to tobacco smoke. Once the variation of natural [THg] is determined, the hair can then be directly exposed to smoking cigarettes and run through the DMA-80 to determine the difference of Hg in the hair. If the difference found to be significantly greater, a separate group (exposed to tobacco smoke) can be treated with the Triton-X detergent used in Gaxiola-Robles This would help determine the affinity of hair with Hg in tobacco smoke, and whether it can be simply washed out by a high strength detergent. The majority of the project will be preparing samples for analyses, as the DMA-80 is autonomous once sample boats have been loaded. Preparing samples would include: acquisition, extraction, freeze drying (if necessary), and homogenization. Over the course of the spring semester, three months would be dedicated to data acquisition, and one month for 6

7 analyzing the results while creating and presenting a paper of my findings. If more time is available, additional ideas will be pursued if presented during research. Anticipated Outcomes: I expect there to be detectable [THg] and [MeHg] in the cigarettes and hair samples. Cigarette [THg] will more than likely fall within the µg/kg of THg (results of summer research) if growing, treating, and cigarette production has been kept constant, between La Paz and Fairbanks. [MeHg] is a complete mystery, as there has been no significant research done in regards to commercial cigarettes. Besides conclusions drawn from this project, I expect to advance my understanding in mercury toxicology, as well as the research process while refining laboratory skills associated with biochemical research. Mentoring: Dr. O Hara will be the faculty mentor; however I will be working more directly with John Harley. There are other technicians and graduate students in the O Hara lab including Andrew Cyr, and Stephanie Kennedy (post-doc students) and Maggie Castellini (a research professional) who will assist our research with methylmercury measurements. The Wildlife Toxicology Lab meets every week to discuss project developments, and agendas, which will prove to be an excellent source for more project guidance. John and I plan to meet twice a week or more depending on our spring schedules, as well as meeting with Dr. O Hara once every two weeks. Dr. O Hara and I will review progress on the project, examine data, and set objectives for the near future. Meetings with John will result in plans for analyses, method discussion, and go over quality assurance quality control data, as well as other miscellaneous help with the project. 7