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Emission of Endocrine Disruptors from Biomass & Diesel Fuel Composition
Impact Statement: Concerns have been raised regarding the possible harmful effects of exposure to certain chemicals that are capable of modulating or disrupting the function of the endocrine system. These chemicals, referred to as endocrine disrupting chemicals (EDCs), have the capability to interfere with the production, release, transport, metabolism, or elimination of the natural hormones in the body responsible for the regulation of developmental processes. Exhaust samples from potential biomass and diesel generator sources have been analyzed to assess potential for emission of EDCs, since combustion sources are known to emit a multitude of compounds whose structure and chemical composition have or are suspected to have EDC-like behavior. The intent of this screening effort was to provide discerning evidence for nominating sources for further EDC characterization. Our intent is to sample and chemically characterize multiple combustion sources to determine whether EDCs are emitted from combustion sources and in what quantity.
Project Description/Current Status :
- Within the last few years, EDCs have been identified in effluents from both pharmaceutical and industrial usages. A number of these compounds are mutagenic and have the ability to form DNA and protein adducts with the ability to activate aryl hydrocarbon receptor (AhR)-mediated activity, or interfere with estrogen receptor (ER)-mediated signaling.
- Recent work suggests that effluent from pulp mills may contain such chemicals, although their origination and identity are unknown. It has been suggested that EDCs are naturally occurring in trees, including phytosterols, such as b-sitosterol, and their degradation byproducts. During bleaching with chlorine dioxide, unsaturated sterols such as b-sitosterol are oxidized, while saturated sterols are unaffected. A portion of the sterols apparently passes through wastewater bio-treatment processes, and recently, it has been reported that some of the sterols cause some of the same responses in fish as observed by exposure to bio-treated whole pulp mill effluent.
- Recent work determined the estrogenic activity of chemicals in diesel exhaust particles and provided an initial identification of EDCs from combustion and vehicular emissions. 4,6-Dimethyldibenzothiophene was identified and confirmed as a compound emitted by diesel engines that possessed estrogenic activity. In addition, another study reported that in utero exposure to diesel exhaust masculinized female rat offspring.
- The overall approach of this research was to conduct analogous bioassay-directed chemical analysis experiments using a cell line stably expressing an androgen-responsive reporter gene and to evaluate its ability to screen compounds responsible for androgenic activity. The technique involved the separation of sample extracts into fractions followed by the determination of their toxicity by incorporating bioassays. The results obtained provided detailed chemical analysis of the toxic fractions produced from the sample.
- The parent cell line for this experiment, MDA-kb2 human breast cancer cells, was chosen for its ability to express high levels of functional, endogenous androgen receptor (AR) and has been stably transfected with androgen responsive MMTV-luc promoter reporter genes.
- In order to identify the individual chemical or chemicals that possibly exhibit androgenic activity in the original source, High-Performance Liquid Chromatography (HPLC) was used as a fractionation technique. The indicated, positive fractions were analyzed by Gas Chromatography/Mass Spectroscopy for chemical identification.
Results:
- Pre-screening the androgen assay with pooled or combined Fireplace (Oakwood/Pinewood and Oakwood/Synthetic Wood) and Woodstove (Oakwood) samples, there was considerable androgenic activity compared to the positive control Dihydrotestestorone (DHT). The most potent androgenic activity was shown in the pooled samples from the Oakwood/Pinewood and Oakwood/Synthetic Wood expressing a maximum 6-fold induction of luciferase activity expressed over media.
- High Performance Liquid Chromatography was employed to isolate the active compounds and Gas Chromatography/Mass Spectroscopy was used to identify seven possible candidate androgenic compounds from the biomass samples. The compounds were identified as by-products of Lignin, with the exception of Caprolactam. The source of Caprolactam was identified in the fraction from pooling fraction Fireplace (Oakwood/Synthetic wood). The by-products of Lignin determined in the chromatogram were Vanillin, Syringaldehyde, 3,4,5,-Trimethoxyacetophenone, 2,4,5-Trimethoxybenzoic acid, 3,4,-Dimethoxy-5-Hydroxybenzaldehyde, and Acetosyringone.
- In order to test the presence of androgenic activity, the seven compounds were evaluated with the MDA-KB2 cells at a 20 mM concentration. The luciferase response from each of the seven standards: Vanillin, Syringaldehyde, 3,4,5,-Trimethoxyacetophenone, 2,4,5-Trimethoxybenzoic acid, 3,4,-Dimethoxy-5-Hydroxybenzaldehyde, Acetosyringone, and Caprolactam did not exhibit ANY SIGNIFICANT considerable androgenic activity compared to the positive control. However, the androgenic activity one of the HPLC fractions may be due to additional structural isomers from one of the compounds that were unidentified in the gas chromatogram.
- We evaluated 1 mL and 0.5 mL extracts from the diesel generator sample with the MDA assay were the results showed a 6-fold luciferase induction over media that was equal to positive control 0.1 nM DHT. The 1 mL dosing of the diesel extract with 1 mM of the anti-androgen hydroxyflutamide also showed a significant reduction in the response of activity to the AR receptor.
We believe that our approach may be useful for screening vehicular and stationary combustion sources for androgenic activity. This study demonstrates the usefulness of bioassays to characterize complex combustion emissions for the purpose of comparing the relative health risks posed by combustion. Bioassy-directed chemical fractionation analysis can be a highly effective way to combine biological and chemical analysis in order to characterize the potentially harmful components of complex combustion emissions. Using the MDA-kb2 cells, we were able to possibly identify androgenic activity from the combustion of biomass only in vitro activity. We were also able to identify possible activity from the diesel extract. Our next step is to identify and confirm the structure of the compound or compounds that present the androgenic activity. Further efforts are still needed in developing new ways of identification of unknown compounds from these complex environmental samples. Only then, quantifying and establishing cause-effect relationships with this bioassay will provide a better understanding.
References:
- Kavlock, R.; Daston, G.; Gray, L.; Kaattari, S.; Environmental Health Perspective. 104 (Supp. 4) p. 715-740, 1996.
- Durant, J.; Busby W.; Lafleur A.; Penman B.; Crespi C., 1996. Mutation Research. 371:123-157.
- Parks, L.; Lambright, C.; Orlando, E.; Guillette, L.; Ankley, G.; Gray, E. Toxicology Sciences, 62, 257-267, 2001.
- Schuetzle, D.; Lewtas, J. Analytical Chemistry, Vol. 58, No. 11, 1996, p. 1060A-1071A.
- Wilson, V.; Bobseine, K., Lambright, C., Gray, E. Toxicology Sciences 66, 69-81, 2002.
Contact Information: For more information concerning combustion processes and endocrine disrupting chemicals, please contact Dr. Clyde V. Owens at phone (919) 541-1133 or fax (919) 541-0290.