Team:Bielefeld-CeBiTec/Project/DateRapeDrugs
Date Rape Drugs
Prevention and Diagnostics
Overview
Date rape drugs are a wide-spread issue. Intoxication can have serious effects on health and emotional state. We established a new, easy to use biosensor which is able to detect possible ingredients of date rape drugs. In combination with our cell free designs, the sensor is applicable for everyone in the open field. The usage is not limited to the testing of beverages, but might also be applicable in diagnostics.
The term date rape drug is referring to sedatives, that may cause unconsciousness, loss of will and ability to resist assault (Jansen and Theron 2006). In addition, a frequent consequence of date rape drugs is memory loss. A chemical often used as date rape drug is GHB, gamma-hydroxy butyrate. While GHB is regulated by law in certain countries, precursors are publicly available. One of these precursors is GBL, gamma-butyrolactone. In Germany and the UK this precursor can be purchased by the general public. After ingestion of GBL, the substance is rapidly converted into GHB and therefore has the same effects (Busardò and Jones 2015). Trace amounts of GHB occur naturally in the human body. It is supposed to function as a precursor of one of the main neurotransmitters, GABA (gamma-aminobutyric acid). GABA is a substance available without any restrictions.
Our biosensor works with a GHB/GBL inducible operon obtained from Agrobacterium tumefaciens on the one hand, and a GABA inducible operon from Bacillus subtilis on the other hand. Our GBL/GHB sensor has been tested in vivo, in our newly established assay PRIA (plasmid repressor interaction assay) and has been proven functional in our CFPS (cell free protein synthesis) assay as well as in cell extract lyophilized on paper.
Background information
Pharmacology and toxicology of GHB/GBL
The maximum amount of GHB in the blood plasma is found 20-40 minutes after ingestion. At this time, the drug reaches its peak effect on behavior. About 100 mg/ L may cause euphoria and disinhibition, while 500 mg/L may have deadly consequences and causes respiratory depression ultimately leading to circulation failure. About 1-5 % of the ingested substance are recoverable in urine. The time window for the detection is unfortunately very small (3-10h) (Busardò and Jones 2015). This decreases the chance of detecting drug intoxications, because cases are often reported too late.
Date rape drugs in numbers
While it is commonly assumed that date rape drugs pose a threat to women, recent studies show an increased portion of male victims. In the emergency department in Barcelona, 505 cases of GHB intoxication were reported during the years 2000-2007, 68% of which were men. The mean age was 24.7 years. Of all patients, 97 % developed symptoms in public places (Galicia et al. 2011). In our area, it was difficult to obtain numbers of cases. No studies or hospital data was available, in part because of data protection reasons. According to the helpline at our university, 3 cases of suspected intoxication with date rape drugs were reported by women at our university in the year 2014, while the local police recorded no cases. This is probably due to the small time window in which the detection of the ingestion is possible. Mrs. Frese, who works at the helpline at our university, estimates the dark figure to be a lot higher, because victims are often frightened and wait too long to seek help. “A test strip would be a valuable tool that could effectively prevent crimes.” Mrs. Frese told us in an interview.
Consequences of intoxication
The consequences of date rape drug intoxication are not only of clinical nature. In an interview with our team, Dr. Jorch, doctor at our local childrens hospital and expert in prevention, explained the dangers of date rape drugs. “Date rape drug intoxication can be a traumatic experience. Not only the possibility of crimes, but also the loss of control and memory are consequences that victims have to face.” Unfortunately, the detection of date rape drugs intoxication is a problem in the daily clinical schedule due to the short time frame. “In case of admittance of an unconscious patient, we usually determine the blood alcohol level first, because the victims are often admitted from a party where alcohol consume is likely." Dr. Jorch explained. "If the results are not congruent with the level of consciousness, further tests are scheduled. But until then, several hours might already have passed. The availability of a cheap, reliable and easy to handle test strip would probably increase the number of tests for date rape drugs, because at the moment we need to send samples to a laboratory. The availability of a test strip for other recreational drugs would also be a valuable tool to quickly determine the ingested toxin and avoid the possible consequences.” He points out that crimes are often committed by people close to the victim. Therefore, the likelihood of testing ones beverage is often not given, as it might have been bought by a person the victim trusts. A test strip could not only be a tool in prevention, but also in diagnostics and forensics.
Detection
Screening for date rape drugs is possible in a specific time frame, 3-10 h after intoxication. Common practice is the detection of GHB in a urine sample via gas spectroscopy. Rapid tests have the disadvantage of cross-reaction with ethanol, which is often involved as well in cases of intoxication (Franken et al. 2015). These enzymatic assays are therefore not suitable for preventional testing of beverages. A repressor/DNA interaction based test has the advantage of specifity. In addition, our cell extract is robust to certain amounts of ethanol. Our rapid test provides significant advantages compared to current testing methods.
Underlying pathways
Catabolism of GBL in A. tumefaciens
The plant pathogen Agrobacterium tumefaciens has been widely used in plant research, as it can transform plant cells with the help of the Ti-plasmid (tumor-inducing plasmid). A. tumefaciens is able to utilize the uncommon carbon and energy source GBL found in plants. To do this, the bacterium uses the enzymes of the blcABC operon (Chai et al. 2007). This operon is localized on the second megaplasmid of A. tumefaciens C58 strain, pAtC58. blcABC expression is controlled by the repressor protein BlcR . When GBL is not present, two dimers of BlcR form a tetramer and inhibit operon expression by binding to an operator sequence localized in front of the operon, thereby hindering the polymerase from transcribing the DNA (Pan et al. 2011, 2013). The inhibition of operon repression was shown to be inducible not only by GBL, but even more so by GHB and SSA (succinic semialdehyde) (Chai et al. 2007).
The enzymes coded for in the blcABC operon catalyze the reaction of GHB to GBL in a first step (Figure). If GBL is taken up by an organism containing the operon, it is processed to GHB, facilitating increased induction. Further, GHB is processed to SSA that finally enters the GABA pathway. Therefore, the operon enables the chassis to utilize GHB or GBL as carbon and nitrogen source. It has been shown that the transformation of the operon enables E. coli to grow on GBL as sole carbon source. (Carlier et al. 2004). Hence, the induction might be increased by the additional transformation of the operon containing blcA in further experiments.
The BlcR tetramer binds the operator site which is overlapping to the blcABC promoter and precents transcription of the operon. In the presence of GBL or GHB, BlcR is inactivated and can not bind to the DNA. The blcABC operon is transcribed.
In principle, our biosensor is based on the repressor BlcR under the control of a constitutive promoter, and the binding sequence of the promoter Pblc. This binding sequence is following an inducible T7promoter. The promoter is followed by a specific translation enhancing 5' untranslated region and controls transcription of sfGFP, which is the output signal. In the absence of GBL or GHB the BlcR repressor binds to its operator sequence and inhibits the transcription of sfGFP. If GBL or GHB are present in the sample solution, BlcR cannot bind its operator sequence anymore. The T7 polymerase is then able to transcribe the sfGFP and a fluorescence signal can be observed. A more detailed description can be found in the result section.
For testing beverages, the biosensor test strip needs to be cell-free to prevent the contamination with genetically modified organisms. Hence, this device is optimized to function in our cell-free protein synthesis (CFPS) assay, but works in vivo as well. For even faster results than in CFPS, we added a his-tag to the repressor BlcR. This aims to obtain a purified repressor for use in our developed plasmid repressor interaction assay (PRIA). While in CFPS proteins still need time to be synthesized, they are added fully functional in PRIA. This optimization enables the quick detection of date rape drugs.
Catabolism of γ-aminobutyric acid (GABA) in B. subtilis
GHB and GBL are both toxic substances for humans which requires special precautions in terms of safety. Therefore we first started with the idea to detect not the toxic substances itself, but similar analogs. The substance GABA ist not toxic for humans and only differs to GHB in the amin group instead of the hydroxy group. The aerobic gram positive bacterium B. subtilis has been analyzed comprehensively. It is able to use GABA in the so called GABA-shunt pathway. This pathway uses the gabTD genes, which are activated by the transcription factor GabR in the presence of GABA and pyridoxal 5’-phosphate (PLP). For this activation, GabR binds the gabT promoter. In addition, gabR underlies an autoregulatory mechanism and binds to its own promoter, repressing the transcription of gabR in presence of PLP and GABA (Edayathumangalam et al. 2013). Because of the structural similarity to GHB, we first designed a biosensor detecting GABA as a metabolite of the natural GHB pathway as a proof of concept as a basis for the GBL/GHB biosensor. During our investigation on GBL and GHB we encountered the problem that we might provide information on the production of these substances to a wider audience. Dealing with the issue of Dual Use , we decided to provide an extensive report on that theme as a part of our Human Practices approach.
References
Busardò, Francesco P.; Jones, Alan W. (2015): GHB Pharmacology and Toxicology: Acute Intoxication, Concentrations in Blood and Urine in Forensic Cases and Treatment of the Withdrawal Syndrome. In: Current neuropharmacology 13 (1), S. 47–70. DOI: 10.2174/1570159X13666141210215423.
Carlier, Aurélien; Chevrot, Romain; Dessaux, Yves; Faure, Denis (2004): The assimilation of gamma-butyrolactone in Agrobacterium tumefaciens C58 interferes with the accumulation of the N-acyl-homoserine lactone signal. In: Molecular plant-microbe interactions : MPMI 17 (9), S. 951–957.
Chai, Yunrong; Tsai, Ching Sung; Cho, Hongbaek; Winans, Stephen C. (2007): Reconstitution of the biochemical activities of the AttJ repressor and the AttK, AttL, and AttM catabolic enzymes of Agrobacterium tumefaciens. In: Journal of bacteriology 189 (9), S. 3674–3679. DOI: 10.1128/JB.01274-06.
Edayathumangalam, Raji; Wu, Rui; Garcia, Roman; Wang, Yuguang; Wang, Wei; Kreinbring, Cheryl A. et al. (2013): Crystal structure of Bacillus subtilis GabR, an autorepressor and transcriptional activator of gabT. In: Proceedings of the National Academy of Sciences of the United States of America 110 (44), S. 17820–17825. DOI: 10.1073/pnas.1315887110.
Franken, Linda G.; Andrews, Louise M.; Slooff, Valerie D.; Wildt, Saskia N. de; Koch, Birgit C. P. (2015): Intoxication of a young girl reveals the pitfalls of GHB rapid screening. In: Therapeutic drug monitoring. DOI: 10.1097/FTD.0000000000000244.
Galicia, Miguel; Nogue, Santiago; Miró, Oscar (2011): Liquid ecstasy intoxication: clinical features of 505 consecutive emergency department patients. In: Emergency medicine journal : EMJ 28 (6), S. 462–466. DOI: 10.1136/emj.2008.068403.
Jansen, Karl L. R.; Theron, Lynn (2006): Ecstasy (MDMA), methamphetamine, and date rape (drug-facilitated sexual assault): a consideration of the issues. In: Journal of psychoactive drugs 38 (1), S. 1–12.
Pan, Yi; Wang, Yi; Fuqua, Clay; Chen, Lingling (2013): In vivo analysis of DNA binding and ligand interaction of BlcR, an IclR-type repressor from Agrobacterium tumefaciens. In: Microbiology (Reading, England) 159 (Pt 4), S. 814–822. DOI: 10.1099/mic.0.065680-0. Pan, Yi; Fiscus, Valena; Meng, Wuyi; Zheng, Zhida; Zhang, Lian-Hui; Fuqua, Clay; Chen, Lingling (2011): The Agrobacterium tumefaciens transcription factor BlcR is regulated via oligomerization. In: The Journal of biological chemistry 286 (23), S. 20431–20440. DOI: 10.1074/jbc.M110.196154.
