Team:Bielefeld-CeBiTec/Project/DateRapeDrugs
Date Rape Drugs
Prevention and Diagnostics
Overview
Date rape drugs are a growing issue in our local area. Intoxication can have serious effects on health and emotional state. We established a new biosensor which is able to detect possible ingredients of date rape drugs to provide a tool for everyone to use. 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, 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 often regulated by law in different countries, precursors are commonly available. One of these precursors is GBL, gamma-butyrolactone. In Germany this precursor is available without restrictions. 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. GABA is a substance available without any restrictions.
Our biosensor works with a GHB/GBL inducable operon obtained from Agrobacterium tumefaciens on the one hand, and a GABA inducable operon obtained 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 cell extract 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. The drug therefore reaches its peak of effect on behavior. About 100 mg/ L may cause euphoria and disinhibition, while 500 mg may have deadly consequences and cause respiratory depression ultimately leading to circulation failure. 1-5 % of the ingested substance are recoverable in urine. The time window of 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 increase of intoxication in men. During the years 2000-2007 in the emergency department in Barcelona, 505 cases of GHB intoxication were reported, 68% of which were men. The mean age was 24.7 years. 97% of patients developed symptoms in public places (Galicia et al. 2011). In our area, it was difficult to obtain numbers of cases, due to the lack of studies and the unavailability of data from hospitals because of data protection reasons. According to the helpline at our university in the year 2014, 3 cases of suspected intoxication with date rape drugs were reported by women at our university, 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.”
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 traumatical experience. Not only the possibility of crimes but the loss of control and memory are consequences that victims have to face.” Unfortunately, the detection of date rape drugs intoxication is also 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. 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 not often 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 therefore has the advantage of specifity. In addition, our cell extract is robust to certain amounts of ethanol. Our rapid test provides significant advantages against current testing methods.
Our sensor
Catabolism of γ-butyrolactone (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 γ-butyrolactone (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 stronger by GHB (gamma-hydroxybutyrate) 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. If GBL is taken up by an organism containing the operon, it is processed to GHB that even stronger induces the operon. Further, GHB is processed to succinic semialdehyde that finally enters the 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, as further experiments, the induction might be increased by the additional transformation of the operon containing blcA.
Therefore, our sensor is basing 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 inducable T7 promoter. The promoter is followed by a 5' untranslated region and controls transcription of sfGFP, which is the output signal. The device can detect both GBL and GHB.
For testing beverages, the biosensor test strip needs to be cell free. Hence, this device is optimized to function in our cell free protein expression system (CFPS), but works in vivo as well. For even faster results than in CFPS, we additionally 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 to be synthesized, they are added fully functional in PRIA. This optimization is a perfect fit fot the quick detection of date rape drugs.
Catabolism of γ-aminobutyric acid (GABA) in B. subtilis
The aerobic gram positive bacterium B. subtilis is comprehensively analyzed. It is able to use y-aminobutyric acid (GABA) in the so called GABA-shunt as carbon and nitrogen source. Therefore, it uses the gabTD genes, that are activated by the transcription factor gabR in 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 additionally designed a biosensor detecting GABA as a metabolite of the natural GHB pathway.
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.