Microscale application of the SOS-LUX-TEST as biosensor for genotoxic agents

Analytica Chimica Acta 387 (1999) 289±296

Microscale application of the SOS-LUX-TEST as biosensor for genotoxic agents Petra Rettberga,*, Christa Baumstark-Khana, Klaus Bandela, Leonid R. Ptitsynb, Gerda Hornecka a

Deutsche Forschungsanstalt fuÈr Luft- und Raumfahrt, Institut fuÈr Luft- und Raumfahrt medizin, Abteilung Strahlenbiologie, 51147, KoÈln, Germany b State Scienti®c Centre of Russian Federation, GNII Genetica, 1,1-st Dorozhny Proezd, 113545, Moscow, Russian Federation Received 21 July 1998; received in revised form 11 December 1998; accepted 14 December 1998

Abstract The bacterial biodetection system SOS-LUX-TEST has been developed for rapid detection of environmental genotoxins. This cellular bioassay is based on the receptor reporter principle with the SOS system as receptor sensitive to DNA damage and the bioluminescence system giving the optical signal which is registered by an appropriate detector. The lux-operon of a marine photobacteria (Photobacterium leiognathi) containing the whole information for the synthesis of the bacterial luciferase and its substrate was cloned downstream of a SOS-controlled promoter and introduced on a plasmid into E. coli. This system reacts in a dose-dependent manner with bioluminescence to any agent like radiation or chemicals which produce damages to the DNA molecules inside these cells. For the measurements of the genotoxic potential of chemical compounds a microplate luminometer was employed which allows continuous incubation and shaking during the registration of the signal. The kinetics of bioluminescence was always monitored over a period of at least 5 h. Typical model compounds with various DNA damaging potencies were tested. # 1999 Elsevier Science B.V. All rights reserved. Keywords: SOS-response; Luminescence analysis; Recombinant bacteria; Biosensor

1. Introduction Cells of various species have been used to evaluate the toxicity and mutagenicity of numerous compounds, many of pharmaceutical interest. Assay systems for identi®cation of mutagenic or carcinogenic agents cover tumour induction in animals [1], the *Corresponding author. Address: DLR, Institute of Aerospace Medicine, Radiation Biology Section, Linder Hoehe, 51147 Cologne, Germany. Tel. +49-2203-601-3594; fax: +49-220361970; e-mail: [email protected]

human somatic mutation test of the HPRT gene in peripheral blood cells [2±5] and induction of chromosome aberrations [6]. Such assays are generally lengthy and expensive, and provide little information on mechanisms of genotoxicity. These constraints have led to the development of a number of short-term test systems designed for detecting potential carcinogens. They are based on the supposition that damage to DNA is the most likely major cause of carcinogenesis [7±9]. In the Ames test several strains of Salmonella have been used to detect various kinds of mutagens. For example, the strains

0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00049-5

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TA97 and TA98 detect short frame shifts and react to such mutagens as ICR-191, 9-aminoacridine and diethylsulphate [10], whereas strain TA100 responses to MNNG, sodium azide and methyl methanesulphonate and detects mutagens that cause base-pair damages while strain TA102 detects oxidative compounds and ionising radiation [11±13]. This test was validated in studies of hundreds of chemicals. Nearly 90% of the carcinogens tested were mutagenic in these studies. In recent years coupling of cells with a suitable transduction signal has led to the development of toxicity tests based on the alteration of a cell metabolic function by the toxic substance under examination. In the SOS-chromotest a special strain of Escherichia coli has been used in which the structural gene for b-galactosidase, lacZ, is under control of sulA, a SOScontrolled gene involved in cell division inhibition [14]. In response to DNA-damaging agents, a set of functions known as SOS-response are induced which include synthesis of a number of proteins such as RecA and UmuC/D proteins related to mutagenesis [15,16]. The SOS-chromotest provides a simple and direct colorimetric assay for b-galactosidase synthesised in response to a genotoxic agent. It detects ionising and UV-radiation with high sensitivity [13,17]. The SOS-induction potency (SOSIP) is closely correlated with the mutagenic potency determined in the Ames mutatest for most of the agents tested [14]. In recent years, a new generation of reagents that report on speci®c molecular events in living cells, the luminescent protein biosensors, has evolved [18±20]. Of the many different strategies available for using as genetic reporters, bioluminescence offers the most nearly ideal situation, because the reporter measurements are nearly almost instantaneous, they are exceptionally sensitive and quantitative, and typically there is no endogenous activity in the host cells to interfere with quanti®cation. Luciferase genes have been cloned from bacteria, beetles (including ®re¯y), Renilla and Aequorea. Of these, only the luciferases from bacteria, ®re¯y and Renilla have found general use as indicators of gene expression [21,22]. Bacterial luciferase is a dimeric enzyme of 80 kDa found predominantly in several marine bacteria [23,24]. The luminescence is generated from an oxi-

dation reaction involving the reduced ¯avin mononucleotide FMNH2 and a long-chain aliphatic aldehyde to yield FMN, carboxylate and blue light of 490 nm. The complete lux-operon consists of the genes encoding the bacterial luciferases luxA and luxB for the  and subunits of the enzyme. [25]. Whereas the luxC, luxD and luxE genes encode the three proteins of the fatty acid reductase complex needed to recycle the reaction product back to the aldehyde substrate. Recycling of the FMN product is achieved through the normal redox homeostasis of the bacterial cells. Thus, autonomous expression of luminescence can be attained in bacterial cells with expression of the lux-operon. All other bioluminescent reporter systems require the exogenous addition of luciferin. A biosensor based on genetically modi®ed bacteria has been developed recently by our group for the purpose of performing a rapid genotoxicity test in aqueous environmental matrices [26,27]. The lux-test is based on the SOS-induction in a similar way as compared with SOS-chromotest. A special E. coli plasmid, pPLS-1, has been constructed in which the promoterless lux-operon (luxCDABE) of Photobacterium leiognathi is under control of the SOS-dependent col promoter and its synthesis is therefore regulated by the SOS-system. DNA damage leads to increased level of luciferase and therefore increased bioluminescence in presence of a variety of mutagens and also after exposure to ionising and UV-radiation. This biosensor has now been adapted to a microscale approach using a microtiter plate luminescence reader. 2. Experimental 2.1. Molecular cloning techniques Construction of the plasmid pPLS-1 (DSM 10333) was performed using standard methods as described [26]. The plasmid carries the luxCDABE genes downstream of a SOS-dependent promoter and thereby has all functions necessary for SOS-inducible bioluminescence. 2.2. Bacterial strain and growth conditions pPLS-1 was used to transform the strain E.coli K12 C600 (Fÿ thi-1 thr-1 leu136 lacYI tonA21 supE44)

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[28]. The recombinant E. coli C600 (pPLS-1) was stable over extended periods of incubation and was used as reporter strain in the SOS-dependent lux-test. Bacteria were grown at 378C in L-medium [29] supplemented with 50 mg/ml ampicillin for positive selection of cells carrying the plasmid pPLS-1. 2.3. Compounds used for genotoxicity using the SOS lux assay The following compounds having different mutagenic power were used for the assay: mitomycin C (MMC) which induces predominantly DNA intrastrand crosslinking; potassium dichromate (K2Cr2O7) which in combination with intracellular glutathione or ascorbate induces apurinic/apyrimidinic sites (APsites) and DNA single strand breaks (SSB) as well as lipid peroxidation. Additionally, chloramphenicol (CAM) was used as negative control. If not otherwise indicated, chemicals were purchased from Sigma. Chemicals used for genotoxicity testing were dissolved in distilled water at high concentrations and diluted for the test. In case of poor solubility of some of the compounds dimethyl sulphoxide (DMSO) was used as a solvent. Opaque 96-well plates with clear bottom (EG&G Berthold, Germany) were prepared in that way, that each well contained 10 ml of either the solvent or different concentration of the test compound. In the test dilutions, overall DMSO concentrations did not exceed 1% (v/v). 2.4. Testing of genotoxicity with the SOS lux assay Bacteria were grown overnight on a rotative shaker at 378C. After dilution (1:50) in fresh L-medium, the culture was incubated at 378C until the absorbance at 570 nm (A570) reached about 0.1±0.3. From this culture 90 ml aliquots were added per well of the prepared microplate, which was placed into the Lucy 1 luminometer (Antos Mikrosysteme GmbH, Krefeld, Germany) and incubated at 308C. Lucy 1 is a multilabel counting instrument, combining a time-resolved photometer and luminometer in one instrument. The plate transport provides a shaking function and the measurement chamber allows temperatures to be adjusted from ambient to 408C in steps of 18C. It is well-known that depending on the photobacterium donor strain, stable bioluminescence in recombinant

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E. coli cells is mostly expressed at very narrow temperature ranges below 308C [30]. Therefore, incubation and measurements were performed at 308C. Kinetics of light emission and simultaneously the cell density (turbidity measured at 570 nm, A570) were measured for up to 300 min at regular intervals (10 min). Light and absorbance measurements were done within 1 s and 0.1 s, respectively. After completing the measurements, the data for absorbance and light output were transferred into an EXCEL macro sheet. Experiments were repeated three times and the standard errors between the experiments were determined. 2.5. Numerical analysis The factor of SOS-induction Fi is calculated according to Eq. (1) from light emission data of the untreated culture (Lux0) and of the samples treated with the genotoxin (Luxi) and cell growth (absorbance) of the untreated culture (A0) and of the treated sample (Ax). Fi ˆ

Luxi A0 : Lux0 Ax

(1)

This correction for cell concentration is necessary, because some genotoxins delay or impair cell growth and thereby in¯uence the total light emission of the culture. For each dose mean values and standard errors of Fi were determined. Fi was plotted as a function of the dose of the genotoxin for low and moderate compound concentrations. Different criteria have been used in SOS tests to evaluate the degree of genotoxicity [31]. From the dose response curves two parameters for description of genotoxicity were derived: 1. the lower limit of detection which is the dose at which the induction factor Fi reaches a value twice that of the background [32], and 2. the genotoxic potential which is the slope of the response curves at low and moderate concentrations. A substance is considered to be genotoxic, if Fi reaches a value of 2 or more at concentrations where absorbances (cell growth) are not impaired.

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3. Results The already described bacterial biodetection assay SOS lux combines the SOS-system indicative for DNA damaging agents as receptor component with the bioluminescence system as a rapid reporter component. We have applied this system for detection of genotoxic agents using a luminometer reading microplates. Mitomycin C (MMC) is known as a genotoxin which predominantly induces DNA intrastrand cross-linking. It was used as an example for a powerful chemical genotoxin. Fig. 1 shows the kinetics of SOS lux induction during incubation of the cells with MMC at 308C. The growth curve of the untreated control, determined from the A570 was not signi®cantly different from the growth curves of the MMC treated samples up to concentrations of 10 ng/ml (Fig. 1(A)). The bioluminescence signal (Fig. 1(B)), as measured as amount of light ¯ashes per second (cps) increased from the baseline level only slightly within the ®rst 60 min of incubation. Afterwards the luminescence increased continuously for several hours leading to a maximal light emission yield at about 300 min after starting the incubation. The simultaneous measurement of cell density and light emission allows correction for cell concentration. This is necessary as inhibition of growth in¯uences the total light emission of the culture. In order to correct for such luminescence in¯uencing growth effects, the SOSinduction factor Fi was calculated (Fig. 1(C)). The high SOS-induction values (Fi, 30±40) identi®es MMC as a powerful genotoxin. The SOS lux test was sensitive to MMC at concentrations as low as 10 ng/ml. Potassium dichromate (K2Cr2O7) is an example for a weak chemical genotoxin. Its kinetics of SOS lux induction is shown in Fig. 2. The growth curve of the untreated control was not signi®cantly different from the growth curves of the treated samples for concentrations up to 10 mg/ml (Fig. 2(A)). The bioluminescence signal (Fig. 1(B)) increased over the untreated control only for the sample with the highest concentration, where the ®rst cytotoxic effects appear. Correction for cytotoxic effects on bioluminescence induction resulted only in a slight increase in Fi (Fig. 1(C)). The SOS-induction values (Fi, 5±6) identi®es K2Cr2O7 as only weak genotoxin.

Fig. 1. Kinetics of SOS lux induction by mitomycin C (MMC), known as powerful genotoxic agent. Exponentially growing cultures of E. coli C600 (pPLS-1) in L-medium at A5700.1 were treated with different concentrations of the compound. Absorbance readings at 570 nm (A570) display cell growth in presence of MMC (A), in parallel SOS-dependent luminescence was measured as light emission (B). Luminescence was corrected for cell growth according to Eq. (1) resulting in the induction factor Fi ranging up to 40 times of the uninduced state (C).

Chloramphenicol (CAP) was used as a cytotoxic compound displaying no genotoxic potency [32]. Although mutagenicity data are not available [32] it is considered as a non-genotoxin. Fig. 3 shows the bioluminescence kinetics for chloramphenicol treatment. The growth curves of the treated samples differ

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Fig. 2. Kinetics of SOS lux induction by potassium dichromate (K2Cr2O7), known as weak genotoxic agent. Exponentially growing cultures of E. coli C600 (pPLS-1) in L-medium at A5700.1 were treated with different concentrations of the compound. Absorbance readings at 570 nm (A570) display cell growth in presence of K2Cr2O7 (A), in parallel SOS-dependent luminescence output is about one order of magnitude less than for MMC (B). Luminescence was corrected for cell growth according to Eq. (1) resulting in the induction factor Fi of up to 6 (C), thus identifying K2Cr2O7 as only weak genotoxin.

considerably from the control sample, proving CAP to be cytotoxic (Fig. 3(A)). The bioluminescence signal (Fig. 3(B)) of the treated samples decreased with increasing concentrations, when compared with the untreated control. Correction for cytotoxic effects on bioluminescence induction con®rmed CAP not to be a

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Fig. 3. Kinetics of SOS lux induction by chloramphenicol (CAP), known as cytotoxic agent. Exponentially growing cultures of E. coli C600 (pPLS-1) in L-medium at A5700.1 were treated with different concentrations of the compound. Absorbance readings at 570 nm (A570) display reduction in cell growth in presence of CAP (A), in parallel SOS-dependent luminescence was markedly reduced in presence of CAP (B). Luminescence was corrected for cell growth according to Eq. (1) resulting in induction factors Fi sloping down from 1.5 to well below 1 (C), thus identifying CAP as a cytotoxic but not genotoxic agent.

genotoxin, as Fi did not signi®cantly exceed a value of 1 (Fig. 3(C)). Fig. 4 gives the dose response curve for impairment of cell growth and induction of the SOS lux test to MMC, K2Cr2O7 and CAP. The cytotoxic potential of the compounds was assessed from the relative absor-

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is given by that dose at which Fi is doubled, is estimated. For MMC and K2Cr2O7 the criteria required for identifying a substance as genotoxic are well met. The lower limit of detection was estimated to be 1.33 ng/ml and 11 mg/ml for MMC and K2Cr2O7, respectively. The genotoxic potential was evaluated from the slope of the induction curves in a linear plot of Fi versus dose. MMC identi®es itself as a compound having a high genotoxic potential (756/ mg mlÿ1), while K2Cr2O7 has only a low genotoxic potential (0.091/mg mlÿ1). 4. Discussion

Fig. 4. Dose effect curve of SOS lux induction for mitomycin C (MMC), potassium dichromate (K2Cr2O7) and chloramphenicol (CAP). The relative cell absorbance Ax/A0 (A) and the induction factor Fi (B) were calculated for data determined after 300 min incubation at 308C. The dotted line is drawn at Fiˆ2. The lower limit of detection was calculated as point of intersection from the linear regression of Fi versus concentration with the dotted line (Fiˆ2) to be 1.33 ng/ml and 11 mg/ml for MMC and K2Cr2O7, respectively.

bance (Ax/A0) which decreased with increasing dose (Fig. 4(A)). The dose response curves illustrating the genotoxic activities of the model compounds are given in Fig. 4(B). The simultaneous measurement of cell concentration and light emission allows for a discrimination between genotoxic and cytotoxic potency of the test substance. If bioluminescence is not induced and the cell growth is comparable to that of the untreated control, the test substance is neither genotoxic nor cytotoxic. If however, bioluminescence and/ or A570 decrease during incubation, then the test suggests the agent in question to be cytotoxic, as it is the case for chloramphenicol. From the dose response curves for luminescence induction versus drug concentration the lower limit of detection, which

Increasing environmental pollution asks for reliable and easy-to-use detector systems for continuous monitoring of contaminations by different chemical compounds and their mixtures. The enormous diversity of genetic responses in living microbes to their environment is an attractive resource for biosensor designs. In particular, there is much interest in microbial sensors for environmental monitoring where toxicity can be ascertained directly by its action on cellular physiology. Genotoxic agents induce a variety of speci®c DNA lesions which may have different implications for cells: 1. the lesion remains unrepaired and leads to cell death, 2. the lesion may be repaired with no further consequences for the cell, or 3. the lesion may induce the error-prone SOS repair pathways including the synthesis of a number of proteins involved in mutagenesis, such as RecA and UmuC/D [16]. The SOS-inducing signal is supposed to consists of single stranded DNA [33] activating the RecA protein, which in turn inactivates the LuxA repressor by promoting its autodigestion reaction. SOS-dependent bacterial test systems make use of the induction of the SOS-response due to the harmful action of DNA damaging agents. In most systems, the SOS-induction potency is determined by a colorimetric assay for b-galactosidase synthesised in response to a genotoxin. The b-galactosidase assay

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requires the addition of substrate and cofactors like 2nitrophenyl b-D-galactopyranoside (ONPG) or ¯uorescein di-b-D-galactopyranoside (FDG) and chloroquine with the consequence that expression of the reporter gene was mainly analysed in cell lysates. This disadvantage can be bypassed by using luminescent reporter assay systems. The lux gene system has been used in several instances as a reporter for investigating gene expression in bacteria. The main advantage is the availability of a real-time, in-vivo and non-disruptive monitoring system. Genetically controlled bacterial luminescent biosensors have been applied for detection of a variety of environmental pollutants [30,34]. Several recombinant bacteria or plants with plasmids carrying the promoterless lux-operon (luxCDABE) originating from Vibrio ®scheri or other marine bacteria ± or only the luxAB genes coding for the bacterial luciferase subunits  and ± under control of agent-speci®c inducible promoters have been developed and used for the detection of aluminium, nickel and selenite [35], mercury [36,37], arsenic and cadmium [38±40] or naphthalene and salicylate [41,42] as well as middle-chain alkanes [43]. Such biosensors are particularly valuable for environmental monitoring of speci®c analytes which are important contaminants of water, waste, soil or air (heavy metals, napthalensulphonates) and which are expected to be found at ¯uctuating concentrations. Recently, we have constructed a bacterial detection system with speci®city for genotoxins by combining the SOS-system indicative for DNA damaging agents as receptor component with the bioluminescence system as a rapid reporter component. As a consequence of exposure to genotoxic agents the intensity of the emitted light is proportional to the concentration of the compound. The SOS lux test is a relatively simple assay system that can be partially or fully automated for routine measurements. The SOS lux test as compared with other bioassays for mutagenicity and/or genotoxicity has a number of practical advantages, especially when the microscale assay system using the microplate luminometer is applied for high throughput studies. The bioluminescent reporter technology allows in vivo analysis. As the light signal is taken from the living cell, the measurement is non-destructive for the test bacteria. The bioluminescence data of the test

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culture can be continuously monitored during the incubation period. This allows to record the whole time kinetics of SOS-induction from the same culture. As a matter of principle the light of one single cell can be detected by use of a sensitive photodetector. The incubation of the bacteria in presence of the test compounds as well as the assessment of light production takes place in the same microplate, thus enabling simultaneous screening of large numbers of samples. Data of the genotoxic potentials are available within less than one work day making possible a high throughput of samples and compounds within a single run. In order to prove the genotoxic effectiveness, a discrimination between genotoxic and cytotoxic potency of the test substance is needed. This has been achieved by simultaneous measurements of the cell concentration. If bioluminescence is not induced and the cell growth is comparable to that of the untreated control, the test substance is neither genotoxic nor cytotoxic. If however, bioluminescence and/or A570 decrease during incubation, then the test suggests the agent in question to be cytotoxic. The values obtained by the SOS lux test for genotoxicity are in good agreement with published toxicity data obtained in vitro. This sensor can thus be considered to provide a valid instrument for the preliminary evaluation of the toxicity of organic compounds or drugs. The SOS lux test cannot substitute for direct measurements of carcinogenic effects in animals or detection of chromosomal aberrations in humans. It can be helpful in predicting where and when such tests should be done. The range of possible applications comprises e.g. on-line monitoring of air contamination, food control. It is an inexpensive assay system having the potential of a wide utilisation in environmental research. 5. Conclusion The SOS-LUX-TEST in the microplate version is a simple, robust and easy-to-perform assay system for the detection of genotoxic agents. It can be performed in commercially available luminometers. Compared to existing assays it has the advantages of being fast and less labor-intensive due to less reaction steps thereby showing a comparable sensitivity. The reac-

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tion kinetics, which are more informative than a single data point after a ®xed reaction time, can be measured without disrupting the cells. Acknowledgements This work was supported by COPERNICUS Grant CIPA CT-94 0122 from the European Commission. The method for the quanti®cation of genotoxic agents as well as the plasmid pPLS-1 itself was patented by the German Aerospace Centre (DLR): (1) German Patent, N 19544374.8, 23.08.96 and (2) German Patent, N 19549417.2-41, 05.06.97.

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