**4. Future applications**

14 Environmental Monitoring

were collected once yearly, for two years (2005-2006), both upstream and downstream of known WWTP discharge sites. They also performed both chemical analysis (targeting E1, E2, EE2, and E3) and bioassays (using BLYES and BLYR). They were able to detect potentially estrogenic compounds at levels statistically different than the field blanks. Levels of E2 equivalents were detected in the nanomolar range. The authors were able to measure estrogenic responses with BLYES but they were not able to detect a seasonal difference in estrogenicity (some chemicals were detected seasonally via chemical analysis but others were not) though it is unclear whether there was no seasonal effect or whether the estrogens

In both studies (Alvarez et al., 2009; Jardim et al., 2011), the expected response with bioassays was lower than the actual response determined with the bioassay. Expected responses are calculated by multiplying a chemical's concentration (determined through chemical analysis) by its potency relative to a reference estrogen, such as E2. It is expected that if all contaminating estrogenic molecules are detected by chemical analysis then the expected responses should match actual bioassay responses. However, it is difficult to anticipate (and therefore target) all possible endocrine-active contaminants that are present in environmental samples. In addition, prediction of the effects of mixtures of chemicals, especially at low concentrations, has proven to be problematic. Moreover, bioassays are likely to detect metabolites of estrogenic chemicals, as long these molecules continue to interact with the human hormone receptor/response element-sensing systems. Given these reasons, it is natural to expect that chemical analysis is unlikely to ever fully predict actual

The androgen-sensing strain of Leskinen et al. (2005) has been used to monitor wastewater before and after treatment in wastewater treatment plants in several cities in Italy (Michelini et al. 2005). It was determined that both samples (pre- and post-treatment) contained chemicals with androgenic activity, however treatment decreased this activity. They determined that approximately 30% of androgenic activity was typically removed but occasionally activity was reduced by 90%. They attributed the decreased activity to the presence of carbon-based filters, which should bind chemicals, thereby removing them from wastewater. This study illustrates the effectiveness of yeast-based bioreporters for the rapid analysis of samples before and after water treatment. It also demonstrates that wastewater treatment does not necessarily remove chemicals associated with potential endocrine

In addition, the strains of Leskinen et al. (2005) (BMAEREluc/ERα, BMAEREluc/ERβ, BMAEREluc/AR, and BMA64/luc) were used to test several lotion samples, as a simulation of using the strains on complex sample matrices. Five of the seven lotion samples demonstrated estrogenic activity, even at dilutions as low as 1:175. The authors attributed this activity to parabens present in the lotions, given that samples with no parabens were not estrogenic but samples with mixtures of parabens were. The authors state that parabens are present in many cosmetic products and are generally considered safe (Soni et al., 2002), despite having been demonstrated to produce an estrogenic response (Routledge et al., 1998) and being present in breast cancer tumors (Darbre et al., 2004). No androgenic activity was

More recently, Svobodova et al. (2009) examined the endocrine disrupting potential of a commercial PCB mixture (Delor 103) and a series of potential PCB degradation metabolites (chlorobenzoic acids and cholorophenols). The authors did not detect any estrogenic activity with any of the chemicals or mixtures tested using bioluminescent yeast, except that 5 mg/L

were detected at such a low concentration that a conclusion cannot be drawn.

bioassay responses.

disrupting activity.

found for any of the samples.

*Saccharomyces cerevisiae*-based bioluminescent bioreporters offer excellent opportunities beyond bacterial bioreporters for rapid analysis of chemicals with human and environmental significance. Expression of the bacterial *lux* cassette in a lower eukaryote offers many opportunities not only for high-throughput screening systems but also bioprocess monitoring, diagnostic applications, fungal gene expression analysis, and *in vivo* sensing of fungal infections (Gupta et al., 2003). Expression in *S. cerevisiae* has led to advances in transferring this system to mammalian cell lines (Close et al., 2010; Patterson et al., 2005).

The advantages for detection of endocrine-disrupting chemicals in water by *S. cerevisiae lux*based bioreporters are numerous including accuracy, ease of use, not expensive, and amenable to automation in performing and collection of data. In addition to screening aqueous samples, BLYES, BLYAS, and BLYR, and other variants described in the literature are useful for Tier I screening as proposed by the EPA, analysis of wastewater influent and effluent, chemical leaching from manufactured products, for example. In fact, the State Environmental Agency of São Paulo (CETESB) in Brazil is considering using the *S. cerevisiae* BLYES bioassay for routine monitoring of surface and ground water samples for the presence of potentially estrogenic substances. Two of the authors (M.E. and G.S.) have begun routine monitoring of wastewater treatment plant effluents from a treatment facility in TN as well as screening 250 water samples across the state of Tennessee in a broad survey.

Ideally, detection of potential endocrine disruptors (or any other chemical of interest) by bioluminescent bioreporter strains would be coupled to remote detection systems for continuous real-time monitoring. Bioluminescent bioreporter integrated circuits fuse reporter cells to an integrated circuit containing a photodetector (e.g. Sayler et al., 2001; Nivens et al., 2004; Sayler et al., 2004). These devices could be distributed in networks and coupled with wireless communications would send signals indicating the presence/absence of chemical contaminants. Roda et al. (2011) have developed a device that couples estrogenor androgen-sensing *S. cerevisiae* expressing firefly bioluminescence to fiber optics with detection by a CCD sensor, yielding a fully functional biosensor. While this device resulted in strains whose detection limit was approximately 10-fold higher than bioassays performed in the lab and was larger than previously reported remote detection systems, it does

Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters 17

Conroy, O., Quanrud, D.M., Ela, W.P., Wicke, D., Lansey, K.E. & Arnold, R.G. (2005). Fate of

Cooper, R.L. & Kavlock, R.J. (1997). Endocrine disruptors and reproductive development: a

Darbre, P.D., Aljarrah, A., Miller, W.R., Coldham, N.G., Sauer, M.J. & Pope, G.S. (2004).

Daunert, S., Barrett, G., Feliciano, J.S., Shetty, R.S., Shrestha, S. & Smith-Spencer, W. (2000).

de Wet, J.R., Wood, K.V., Helinski, D.R. & Deluca, M. (1985). Cloning of firefly luciferase

Eldridge, M.L., Sanseverino, J., Layton, A.C., Easter, J.P., Schultz, T.W. & Sayler, G.S. (2007).

Fang, H., Tong, W.D., Perkins, R., Soto, A.M., Prechtl, N.V. & Sheehan, D.M. (2000).

Focazio, M.J., Kolpin, D.W., Barnes, K.K., Furlong, E.T., Meyer, M.T., Zaugg, S.D., Barber,

Fossi, M.C. & Marsili, L. (2003). Effects of endocrine disruptors in aquatic mammals. *Pure* 

Gaido, K.W., Leonard, L.S., Lovell, S., Gould, J.C., Babai, D., Portier, C.J. & McDonnell, D.P.

Gros, M., Petrovic, M. & Barcelo, D. (2009). Tracing pharmaceutical residues of different

drinking water sources. *Science of the Total Environment* 402: 201-216. Folmar, L.C., Hemmer, M.J., Denslow, N.D., Kroll, K., Chen, J., Cheek, A., Richman, H.,

vivo and in vitro. *Aquatic Toxicology* 60: 101-110.

library searching. *Analytical Chemistry* 81: 898-912.

*and Applied Chemistry* 75: 2235-2247.

*Pharmacology* 143: 205-212.

genetic-analysis of functions from *Vibrio fischeri*. *Cell* 32: 773-781.

*National Academy of Sciences of the United States of America* 82: 7870-7873. DiGrazia, P., King, J., Blackburn, J., Applegate, B., Bienkowski, P., Hilton, B. & Sayler, G.

*United States of America-Biological Sciences* 80: 120-123.

with reporter genes. *Chemical Reviews* 100: 2705-2738.

slurry reactor. *Biodegradation* 2: 81-91.

*Health Perspectives* 108: 723-729.

24: 5-13.

treatment. *Environmental Science & Technology* 39: 2287-2293.

weight-of-evidence overview. *Journal of Endocrinology* 152: 159-166.

synthetic oligonucleotide probe. *Proceedings of the National Academy of Sciences of the* 

wastewater effluent hER-agonists and hER-antagonists during soil aquifer

Concentrations of parabens in human breast tumours. *Journal of Applied Toxicology*

Genetically engineered whole-cell sensing systems: coupling biological recognition

cDNA and the expression of active luciferase in *Escherichia coli*. *Proceedings of the* 

(1991). Dynamic response of naphthalene biodegradation in a continuous flow soil

*Saccharomyces cerevisiae* BLYAS, a new bioluminescent bioreporter for detection of androgenic compounds. *Applied and Environmental Microbiology* 73: 6012-6018. Engebrecht, J., Nealson, K. & Silverman, M. (1983). Bacterial bioluminescence - isolation and

Quantitative comparisons of *in vitro* assays for estrogenic activities. *Environmental* 

L.B. & Thurman, M.E. (2008). A national reconnaissance for pharmaceuticals and other organic wastewater contaminants in the United States - II) Untreated

Meredith, H. & Grau, E.G. (2002). A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in

(1997). Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. *Toxicology and Applied* 

therapeutic classes in environmental waters by using liquid chromatography/quadrupole-linear ion trap mass spectrometry and automated

demonstrate this device's usefulness in future environmental monitoring. Remotely deployed devices may allow long integration times to account for chronic exposure to lowlevels (ppb) of a potential endocrine disrupter that may not be captured in a single grab sample.

#### **5. Acknowledgments**

The authors would like to thank the Center for Environmental Biotechnology and the São Paulo Research Foundation (FAPESP) for support of our work (FAPESP 2007/58449-2).

#### **6. References**


demonstrate this device's usefulness in future environmental monitoring. Remotely deployed devices may allow long integration times to account for chronic exposure to lowlevels (ppb) of a potential endocrine disrupter that may not be captured in a single grab

The authors would like to thank the Center for Environmental Biotechnology and the São Paulo Research Foundation (FAPESP) for support of our work (FAPESP 2007/58449-2).

Alvarez, D.A., Cranor, W.L., Perkins, S.D., Schroeder, V.L., Iwanowicz, L.R., Clark, R.C.,

Bakhrat, A., Eltzov, E., Finkelstein, Y., Marks, R.S. & Raveh, D. (2011). UV and arsenate

Beresford, N., Routledge, E.J., Harris, C.A. & Sumpter, J.P. (2000). Issues arising when

Bergamasco, A.M., Eldridge, M.L., Sanseverino, J., Sodré, F.F., Montagner, C.C., Pescara,

Bovee, T.F.H., Helsdingen, R.J.R., Hamers, A.R.M., van Duursen, M.B.M., Nielen, M.W.F. &

Bovee, T.F.H., Helsdingen, R.J.R., Koks, P.D., Kuiper, H.A., Hoogenboom, R. & Keijer, J.

Cargouet, M., Perdiz, D. & Levi, Y. (2007). Evaluation of the estrogenic potential of river and

Cespedes, R., Lacorte, S., Raldua, D., Ginebreda, A., Barcelo, D. & Pina, B. (2005).

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. & Prasher, D.C. (1994). Green fluorescent

Close, D.M., Patterson, S.S., Ripp, S., Baek, S.J., Sanseverino, J. & Sayler, G.S. (2010).

Cohn, D.H., Ogden, R.C., Abelson, J.N., Baldwin, T.O., Nealson, K.H., Simon, M.I. &

Guy, C.P., Pinkney, A.E., Blazer, V.S. & Mullican, J.E. (2009). Reproductive health of bass in the Potomac, USA, drainage: part 2. Seasonal occurrence of persistent and emerging organic contaminants. *Environmental Toxicology and Chemistry* 28: 1084-

toxicity: a specific and sensitive yeast bioluminescence assay. *Cell Biology and* 

interpreting results from an in vitro assay for estrogenic activity. *Toxicology and* 

I.C., Jardim, W.D. & Umbuzeiro, G.D. Bioluminescent yeast estrogen assay (BLYES) as a promising tool to monitor surface and drinking water for estrogenicity. *Journal* 

Hoogenboom, R. (2007). A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists. *Analytical and Bioanalytical Chemistry*

(2004). Development of a rapid yeast estrogen bioassay, based on the expression of

treated waters in the Paris area (France) using in vivo and in vitro assays.

Distribution of endocrine disruptors in the Llobregat River basin (Catalonia, NE

Autonomous bioluminescent expression of the bacterial luciferase gene cassette

Mileham, A.J. (1983). Cloning of the *Vibrio harveyi* luciferase genes - use of a

sample.

**5. Acknowledgments** 

**6. References** 

1095.

*Toxicology* 27: 227-236.

389: 1549-1558.

*Applied Pharmacology* 162: 22-33.

*of Environmental Monitoring* (in press).

green fluorescent protein. *Gene* 325: 187-200.

(*lux*) in a mammalian cell line. *Plos One* 5(8).

Spain). *Chemosphere* 61: 1710-1719.

*Ecotoxicology and Environmental Safety* 67(1): 149-156.

protein as a marker for gene-expression. *Science* 263: 802-805.

synthetic oligonucleotide probe. *Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences* 80: 120-123.


Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters 19

Kidd, K. A., Blanchfield, P. J., Mills, K. H., Palace, V. P., Evans, R. E., Lazorchak, J. M., &

estrogen. *Proceeding of the National Academy of Science*, USA, 104:8897–8901. King, J.M.H., Digrazia, P.M., Applegate, B., Burlage, R., Sanseverino, J., Dunbar, P., Larimer,

Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B. &

Korach, K.S., Sarver, P., Chae, K., McLachlan, J.A. & McKinney, J.D. (1988). Estrogen

Kuster, M., Azevedo, D.A., de Alda, M.J.L., Neto, F.R.A. & Barcelo, D. (2009). Analysis of

Layton, A.C., Gregory, B.W., Seward, J.R., Schultz, T.W. & Sayler, G.S. (2000). Mineralization

Le Guevel, R. & Pakdel, F. (2001). Streamlined beta-galactosidase assay for analysis of recombinant yeast response to estrogens. *Biotechniques* 30: 1000-1004. Leskinen, P., Hilscherova, K., Sidlova, T., Kiviranta, H., Pessala, P., Salo, S., Verta, M. &

Leskinen, P., Michelini, E., Picard, D., Karp, M. & Virta, M. (2005). Bioluminescent yeast

Ma, M., Rao, K.F. & Wang, Z.J. (2007). Occurrence of estrogenic effects in sewage and industrial wastewaters in Beijing, China. *Environmental Pollution* 147: 331-336. Meighen, E.A. & Dunlap, P.V. (1993). Physiological, biochemical and genetic-control of

Michelini, E., Leskinen, P., Virta, M., Karp, M. & Roda, A. (2005). A new recombinant cell-

Miller, C.A., Martinat, M.A. & Hyman, L.E. (1998). Assessment of aryl hydrocarbon receptor

O'Connor, J.C., Cook, J.C., Marty, M.S., Davis, L.G., Kaplan, A.M. & Carney, E.W. (2002).

compounds (EACs). *Critical Reviews in Toxicology* 32: 521-549.

naphthalene exposure and biodegradation. *Science* 249: 778-781.

restricted structural probes. *Molecular Pharmacology* 33: 120-126.

Janeiro (Brazil). *Environment International* 35: 997-1003.

USA. *Environmental Science & Technology* 34: 3925-3931.

yeast. *Biosensors & Bioelectronics* 23: 1850-1855.

*Chemosphere* 61: 259-266.

Academic Press Ltd. 34: 1-67.

*Biosensors & Bioelectronics* 20: 2261-2267.

*Journal of Applied Microbiology* 96:33-46.

*Science & Technology* 36: 1202-1211.

Flick, R. W. (2007). Collapse of a fish population after exposure to a synthetic

F. & Sayler, G.S. (1990). Rapid, sensitive bioluminescent reporter technology for

Buxton, H.T. (2002). Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999-2000: A national reconnaissance. *Environmental* 

receptor-binding activity of polychlorinated hydroxybiphenyls - conformationally

phytoestrogens, progestogens and estrogens in environmental waters from Rio de

of steroidal hormones by biosolids in wastewater treatment systems in Tennessee

Virta, M. (2008). Detecting AhR ligands in sediments using bioluminescent reporter

assays for detecting estrogenic and androgenic activity in different matrices.

bacterial bioluminescence. In *Advances in Microbial Physiology, Vol 34*. London,

based bioluminescent assay for sensitive androgen-like compound detection.

complex interactions using pBEVY plasmids: expression vectors with bi-directional promoters for use in *Saccharomyces cerevisiae*. *Nucleic Acids Research* 26: 3577-3583. Nivens, D.E., McKnight, T.E., Moser, S.A., Osbourn, S.J., Simpson, M.L., & Sayler, G.S.

(2004). Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring.

Evaluation of Tier I screening approaches for detecting endocrine-active


Guillette, L.J., Brock, J.W., Rooney, A.A. & Woodward, A.R. (1999). Serum concentrations of

Gupta, R.K., Patterson, S.S., Ripp, S., Simpson, M.L. & Sayler, G.S. (2003). Expression of the

Hakkila, K., Maksimow, M., Karp, M. & Virta, M. (2002). Reporter genes *lucFF*, *luxCDABE*,

Hayes, T.B., V. Khoury, A. Narayan, M. Nazir, A. Park, t. Brown, L. Adame, E. Chan, D.

Hein, R. & Tsien, R.Y. (1996). Engineering green fluorescent protein for improved

Heitzer, A., Malachowsky, K., Thonnard, J.E., Bienkowski, P.R., White, D.C. & Sayler, G.S.

Heitzer, A., Webb, O.F., Thonnard, J.E. & Sayler, G.S. (1992). Specific and quantitative

Jaio, B.W., Yeung, E.K.C., Chan, C.B. & Cheng, C.H.K. (2008). Establishment of a transgenic

activity of malachite green. *Journal of Cellular Biochemistry* 105: 1399-1409. Jardim, W., Montagner, C., Pescara, I., Umbuzeiro, G., Bergamasco, A., Eldridge, M. &

Johnson, F.H., Gershman, L.C., Waters, J.R., Reynolds, G.T., Saiga, Y. & Shimomura, O.

Kavlock, R.J., Daston, G.P., DeRosa, C., FennerCrisp, P., Gray, L.E., Kaattari, S., Lucier, G.,

Kendall, J.M. & Badminton, M.N. (1998). *Aequorea victoria* bioluminescence moves into an

drinking water. *Separation and Purification Technology*. *In press.* 

*Aequorea*. *Journal of Cellular and Comparative Physiology* 60: 85-103.

a review. *Journal of Microbiological Methods* 49: 103-119.

exciting new era. *Trends in Biotechnology* 16: 216-224.

bacterium. *Applied and Environmental Microbiology* 60: 1487-1494.

*Environmental Contamination and Toxicology* 36: 447-455.

*the National Academy of Science*, USA, 107:4612-4617.

molecules. *Genes & Development* 15: 1593-1612.

*Fems Yeast Research* 4: 305-313.

*Biology* 6: 178-182.

*Analytical Biochemistry* 301: 235-242.

various environmental contaminants and their relationship to sex steroid concentrations and phallus size in juvenile American alligators. *Archives of* 

*Photorhabdus luminescens lux* genes (*luxA*, *B*, *C*, *D*, and *E*) in *Saccharomyces cerevisiae*.

*gfp*, and *dsred* have different characteristics in whole-cell bacterial sensors.

Bucholz, T. Stueve, & S. Gallipeau. (2010). Atrazine induces complete feminization and chemical castration in make African clawed frogs (*Xenopus laevis*). *Proceeding of* 

brightness, longer wavelengths and fluorescence resonance energy transfer. *Current* 

(1994). Optical biosensor for environmental online monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter

assessment of naphthalene and salicylate bioavailability by using a bioluminescent catabolic reporter bacterium. *Applied and Environmental Microbiology* 58: 1839-1846. Hellen, C.U.T. & Sarnow, P. (2001). Internal ribosome entry sites in eukaryotic mRNA

yeast screening system for estrogenicity and identification of the anti-estrogenic

Sodre, F. (2011). An intergrated approach to evaluate emerging contaminants in

(1962). Quantum efficiency of *Cypridina* luminescence, with a note on that of

Luster, M., Mac, M.J., Maczka, C., Miller, R., Moore, J., Rolland, R., Scott, G., Sheehan, D.M., Sinks, T. & Tilson, H.A. (1996). Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the US EPA-sponsored workshop. *Environmental Health Perspectives* 104: 715-740. Keane, A., Phoenix, P., Ghoshal, S. & Lau, P.C.K. (2002). Exposing culprit organic pollutants:


Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters 21

Sayler, G.S., Ripp, S., Nivens, D., & Simpson, M. (2001). Bioluminescent Bioreporter

Sayler, G.S., Simpson, M.L., & Cox, C.D. (2004). Emerging foundations: nano-engineering

Schultz, T.W. (2002). Estrogenicity of biphenylols: activity in the yeast gene activation assay.

Schultz, T.W., Kraut, D.H., Sayler, G.S. & Layton, A.C. (1998). Estrogenicity of selected

Schultz, T.W. & Sinks, G.D. (2002). Xenoestrogenic gene exression: structural features of

Schultz, T.W., Sinks, G.D. & Cronin, M.T.D. (2002). Structure-activity relationships for gene

Shimomura, O., Johnson, F.H. & Saiga, Y. (1962). Extraction, purification and properties of

Sohoni, P., Lefevre, P.A., Ashby, J. & Sumpter, J.P. (2001). Possible androgenic/anti-

Soni, M.G., Taylor, S.L., Greenberg, N.A. & Burdock, G.A. (2002). Evaluation of the health

Sonne, C., Leifsson, P.S., Dietz, R., Born, E.W., Letcher, R.J., Hyldstrup, L., Riget, F.F.,

Stoker, T.E., Gibson, E.K. & Zorrilla, L.M. (2010). Triclosan exposure modulates estrogendependent responses in the female Wistar rat. *Toxicological Sciences* 117: 45-53. Svobodova, K. & Cajthaml, T. (2010). New in vitro reporter gene bioassays for screening of

Svobodova, K., Plackova, M., Novotna, V. & Cajthaml, T. (2009). Estrogenic and androgenic

Szittner, R. & Meighen, E. (1990). Nucleotide-sequence, expression, and properties of

Thomas, K.V., Hurst, M.R., Matthiessen, P., McHugh, M., Smith, A. & Waldock, M.J. (2002).

*Journal of Cellular and Comparative Physiology* 59: 223-239.

*Bulletin of Environmental Contamination and Toxicology* 68: 332-338.

*Journal of Environmental Biotechnology* 1: 33-39.

*Microbiology* 7:267-273.

*Chemistry* 17: 1727-1729.

*Toxicology* 40: 1335-1373.

*Biotechnology* 88: 839-847.

*Chemistry* 265: 16581-16587.

*and Chemistry* 21: 1456-1461.

*Science & Technology* 40: 5668-5674.

*Environmental Toxicology* 17: 14-23.

783-786.

173-178.

5925.

Integrated Circuits: Sensing Analytes and Organisms with Living Microorganisms.

and bio-microelectronics for environmental biotechnology. *Current Opinion in* 

biphenyls evaluated using a recombinant yeast assay. *Environmental Toxicology and* 

active polycyclic aromatic hydrocarbons. *Environmental Toxicology and Chemistry* 21:

activation oestrogenicity: Evaluation of a diverse set of aromatic chemicals.

aequorin, a bioluminescent protein from luminous hydromedusan, *Aequorea*.

androgenic activity of the insecticide fenitrothion. *Journal of Applied Toxicology* 21:

aspects of methyl paraben: a review of the published literature. *Food and Chemical* 

Kirkegaard, M. & Muir, D.C.G. (2006). Xenoendocrine pollutants may reduce size of sexual organs in East Greenland polar bears (*Ursus maritimus*). *Environmental* 

hormonal active compounds in the environment. *Applied Microbiology and* 

activity of PCBs, their chlorinated metabolites and other endocrine disruptors estimated with two in vitro yeast assays. *Science of the Total Environment* 407: 5921-

luciferase coded by *lux* genes from a terrestrial bacterium. *Journal of Biological* 

An assessment of in vitro androgenic activity and the identification of environmental androgens in United Kingdom estuaries. *Environmental Toxicology* 


OECD (2002). Appraisal of test methods for sex-hormone disrupting chemicals. Detailed

Patterson, S.S., Dionisi, H.M., Gupta, R.K. & Sayler, G.S. (2005). Codon optimization of

Purvis, I.J., Chotai, D., Dykes, C.W., Lubahn, D.B., French, F.S., Wilson, E.M. & Hobden,

Raman, D.R., Williams, E.L., Layton, A.C., Burns, R.T., Easter, J.P., Daugherty, A.S., Mullen,

Reemtsma, T., Weiss, S., Mueller, J., Petrovic, M., Gonzalez, S., Barcelo, D., Ventura, F. &

Rehmann, K., Schramm, K.W. & Kettrup, A.A. (1999). Applicability of a yeast oestrogen

Ripp, S., DiClaudio-Eldridge, M.L. & Sayler, G.S. (2010). Biosensors as environmental

Roda, A., Cevenini, L., Michelini, E. & Branchini, B.R. (2011). A portable bioluminescence

Ropstad, E., Oskam, I.C., Lyche, J.L., Larsen, H.J., Lie, E., Haave, M., Dahl, E., Wiger, R. &

Routledge, E.J., Parker, J., Odum, J., Ashby, J. & Sumpter, J.P. (1998). Some alkyl hydroxy

Routledge, E.J. & Sumpter, J.P. (1996). Estrogenic activity of surfactants and some of their

Sanseverino, J., Eldridge, M.L., Layton, A.C., Easter, J.P., Yarbrough, J., Schultz, T.W. &

bioluminescent yeast bioreporters. *Toxicological Sciences* 107: 122-134. Sanseverino, J., Gupta, R.K., Layton, A.C., Patterson, S.S., Ripp, S.A., Saidak, L., Simpson,

compounds. *Applied and Environmental Microbiology* 71: 4455-4460.

Organization for Economic Co-operation and Development (OECD). Owens, C.V., Lambright, C., Bobseine, K., Ryan, B., Gray, L.E., Gullett, B.K. & Wilson, V.S.

*Technology* 41: 8506-8511.

*Gene* 106(1): 35-42.

*Chemosphere* 38: 3303-3312.

*a-Current Issues* 69: 53-76.

*Pharmacology* 153: 12-19.

*Toxicology and Chemistry* 15: 241-248.

Hoboken, NJ, Wiley-Blackwell: 213-233.

5458.

26: 3647-3653.

*Microbiology & Biotechnology* 32: 115-123.

*Environmental Science & Technology* 38: 3567-3573.

Review Paper. Series on testing andassessments. No. 21. Paris, France,

(2007). Identification of estrogenic compounds emitted from the combustion of computer printed circuit boards in electronic waste. *Environmental Science &* 

bacterial luciferase (*lux*) for expression in mammalian cells. *Journal of Industrial* 

A.N. (1991). An androgen-inducible expression system for *Saccharomyces cerevisiae*.

M.D. & Sayler, G.S. (2004). Estrogen content of dairy and swine wastes.

Knepper, T.P. (2006). Polar pollutants entry into the water cycle by municipal wastewater: a European perspective. *Environmental Science & Technology* 40: 5451-

screen for the detection of oestrogen-like activities in environmental samples.

monitors. In *Environmental Microbiology, 2nd Edition*. R. Mitchell and J. D. Gu.

engineered cell-based biosensor for on-site applications. *Biosensors & Bioelectronics*

Skaare, J.U. (2006). Endocrine disruption induced by organochlorines (OCs): field studies and experimental models. *Journal of Toxicology and Environmental Health-Part* 

benzoate preservatives (parabens) are estrogenic. *Toxicology and Applied* 

degradation products assessed using a recombinant yeast screen. *Environmental* 

Sayler, G.S. (2009). Screening of potentially hormonally active chemicals using

M.L., Schultz, T.W. & Sayler, G.S. (2005). Use of *Saccharomyces cerevisiae* BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic


**2** 

G.P. Petrova

*Russia* 

*Lomonosov Moscow State University,* 

**Physical Mechanisms of "Poisoning" the Living Organism by Heavy Metals** 

The toxic affect of heavy metals over the living organisms is known to arouse from the alternation of the course for biological reactions in cells. One of such violations appears to be the process of supra-molecular structures formation, for example, the dipole protein nanoclusters in blood. This phenomenon can be well studied in the biological solutions, such as

In the works devoted to problems of pollution of the surrounding environment and ecological monitoring, for today to heavy metals carry more than 40 metals of periodic system D.I. Mendeleyev with nuclear weight over 50 nuclear units: V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd, Sn, Hg, Pb, Bi, etc. Thus the important role in categorize heavy metals is played by following conditions: their high toxicity for live organisms in rather low concentration, and also ability to bioaccumulation. Almost all metals getting under this definition (except for lead, mercury, cadmium and the bismuth which biological role currently is not clear), actively participate in biological processes, are a part some many enzymes. On N.Rejmersa's classification, heavy it is necessary to consider metals with density more than 8 g/cm3 .Thus, heavy metals concern Pb,

The results of our investigations can to conclude that the process of cluster formation

In our works the interaction of some proteins – albumins, globulins, collagen, lisozym, collagenase, creatin cenase, pepsin with heavy metal ions like Cs, Rb, Cu, Cd, Pb in water

Especial influence on some protein like albumins, globulins, collagen, lisozym and so on has the potassium. K+ ions presence in the protein solutions also induced appearance of dipole

The appearance of cluster cans disturbance metabolic processes in the cells, membranes,

The interaction of proteins with ions of alkaline heavy metals like Cs+, Rb+, and Cu2+, Cd2+, Pb2+ in aqueous solutions was studied in our earlier works [4-9] with the Rayleigh-Debye light scattering (RDLS), the photon correlation spectroscopy (PCS) and the fluorescence polarization (FP) methods. It should be noted, that the nano-sized cluster formation process was also registered in some proteins and enzymes solutions like albumin, globulin, collagen, lysozyme, collagenase and so on in the presence of K+ ions which does not belong to the

blood serum, or a widely adopted normal saline solution of albumin [1-3].

**1. Introduction** 

Cu, Zn, Ni, Cd, Co, Sb, Sn, Bi, Hg.

solutions was studied.

protein nano-clusters.

heavy metals group [8, 11].

tissue.

depends on the value of ionic radius metal.

