Genotoxic effect of 2,2’-bis(bicyclo[2.2.1] heptane) on bacterial cells

The toxic effect of strained hydrocarbon 2,2'—bis (bicyclo[2.2.1]heptane) (BBH) was studied using whole-cell bacterial lux-biosensors based on Escherichia coli cells in which luciferase genes are transcriptionally fused with stress-inducible promoters. It was shown that BBH has the genotoxic effect causing bacterial SOS response however no alkylating effect has been revealed. In addition to DNA damage, there is an oxidative effect causing the response of OxyR/S and SoxR/S regulons. The most sensitive to BBH lux-biosensor was E. coli pSoxS-lux which reacts to the appearance of superoxide anion radicals in the cell. It is assumed that the oxidation of BBH leads to the generation of reactive oxygen species, which provide the main contribution to the genotoxicity of this substance.


Introduction
Strained hydrocarbons like norbornane and its non-saturated derivatives are commonly used in the production of rubber, epoxides, medicinal compounds and perfumes [1,2]. Notably, thermotechnical characteristics of strained hydrocarbons, which have extra internal energy due to deformation of valence bond angles, made them attractive for high-performance combustion applications [3]. Strained 2,2'-bis(bicyclo[2.2.1]heptane) (BBH) compound is a promising as a fuel component for liquid rocket engines. It is assumed that BBH is comparable with the unsymmetrical dimethylhydrazine (UDMH) in terms of specific impulse efficiency, but can be significantly less toxic to the environment and personnel working with rocketry. For the purpose of this study, BBH which consists of two strained structures made of 14 carbons and 22 hydrogens (Fig 1) was synthesized from 5-vinyl-2-norbornene [4][5][6]. Here we present the data describing the toxicity of BBH, which we had obtained by utilizing bacterial lux-biosensors. Lux-biosensors are living Escherichia coli cells transformed with hybrid plasmids, containing luxCDABE genes of Photorhabdus luminescens under control of various stress promoters, responsible for increasing the cells luminescence in occurrence of toxicants in the  [7][8][9][10][11]. Similarly designed study of UDMH toxicity has been completed earlier [12,13]. The purpose of this work was to test genotoxicity of BBH and to investigate the mechanisms of genotoxicity if any occurs. The mechanism of BBH genotoxicity was evaluated using specific lux-biosensors of E. coli MG1655 cells with hybrid plasmids pAlkA-lux, pOxyR-lux, pSoxSlux, and pColD-lux, reacting to DNA alkylation, oxidative damage by hydrogen peroxide, superoxide anion radicals and DNA damages that cause an SOS response, respectively. Threshold concentrations of BBH and UDMH were compared for different stress-responsive promoters.

Media and culturing conditions
Cell cultures were prepared by overnight cultivation at 37˚C with aeration at 200 rpm in the 25 ml glass culture tubes in 5 ml of LB media supplemented with 100 μg/ml ampicillin, then diluted 1:100 in LB and grown at 37˚C till reaching OD 600 = 0.1-0.2, which corresponds to early logarithmic phase. The resulting cultures were used for further experiments and luminescence measurements.

Experiment conditions and luminescence measurement
Bacterial cultures were divided into the 200-μl portions in separate wells of 96-well plate, and then 20 μl of tested compound (control toxicant or BBH in concentrations of 100, 10, or 1 mg/ ml) were added. Then cells were incubated without shaking at room temperature with repetitive direct measurements of total bioluminescence (in RLU, relative light units) using plate luminometer LM-01A (Immunotech, Czech Republic). The measurement time depended on the type of biosensor used. The average common measurement time is 2 hours [10,12,13].
Since BBH is a new compound and has an unknown mechanism of interaction with cellular structures, preliminary experiments were extended by 2-3 hours to enable postponed effects detection. After characteristic response time was evaluated series of experiments were conducted with measurement times allowing to see the most significant biosensors signals. This time was 3 hours for pAlkA-lux, 2 hours for pOxyR-lux, 5 hours for pColD-lux, and pSoxSlux. For threshold concentrations identification experiments with each biosensor were carried out with serial twofold BBH dilutions from 1 to 500 mM.

Statistical analysis
Average values and standard deviations were calculated for five independent replications in each experiment. One-way ANOVA followed by Dunnett's multiple comparisons test and one-sample t-test for each concentration of test substance for all biosensors were performed using GraphPad Prism version 8.01 for Windows, GraphPad Software, La Jolla California USA, (www.graphpad.com). Area under curve (AUC) obtained by integrating kinetic curves data.

Induction of PalkA
At the first stage, the ability of BBH to alkylate DNA was investigated with use of E. coli MG1655 (pAlkA-lux) biosensor (Fig 2). The pAlkA-lux plasmid contains lux-genes under the control PalkA, therefore when alkylating agents occur in the sample the luminescence increases. Methyl methanesulfonate (MMS) in concentration of 100 μM was used as the alkylating substance in a positive control. As one can see in Fig 2, none of the tested concentrations of BBH caused an alkylating effect as luminescence does not increase. One-way ANOVA for 1 and 10 g / l BBH and control curves gives a p-value of 0.35. At a maximum concentration of 100 g/l (10%) BBH has a cytotoxic effect, leading to a slight but still statistically significant decrease (p-value < 0.001, Dunnett's test) in the background luminescence of cells (about 2-3 times). BBH causes induction of the oxyR gene promoter (Fig 3). PoxyR activates in response to appearance of hydrogen peroxide or alkyl hydroperoxides. These toxicants can cause modifications of DNA nitrogenous bases, leading to an increase in the rate of mutagenesis [19,20]. The maximum effect is achieved at BBH 1% (Fig 3, curve BBH-2), a lower concentration does  not cause a significant increase in luminescence, and a higher has a general toxic effect on cells, leading to a decrease in the base level of luminescence. One-way ANOVA for BBH (all concentrations) and control curves gives p-value < 0.001. In the presence of catalase, the induction of the PoxyR by 1% BBH was almost abolished. As can be seen from the data shown in the Fig 4, the addition of BBH in concentrations of 10% and 1% leads to the high level of DNA damage, which causes an SOS response. The maximum possible response amplitude of the E. coli MG1655 (pColD-lux) biosensor is about three orders of magnitude [10]. In this experiment, we see the maximum biosensor activation of about 20 times during incubation with BBH at a concentration of 10 g/l. One-way ANOVA for BBH (all concentrations) and control curves gives p-value <0.001.

Induction of PsoxS
Then the appearance of a superoxide anion radical in cells during incubation in the presence of BBH was investigated. For this purpose, lux-genes under the control of the PsoxS promoter were used. Fig 5 shows the luminescence kinetics of E. coli MG1655 (pSoxS-lux) cells incubated with BBH. As a standard inductor for PsoxS promoter paraquat is usually used-a substance that leads to occurrence of superoxide anion radical as a result of reactions with quinones of the respiratory chain. Hydrogen peroxide does not directly react with the SoxR protein but causes lipid peroxidation and the respiratory chain proteins damage that eventually leads to an increase in the superoxide anion radical pool in the cell [10,21]. Thus, H 2 O 2 can also be used in a positive control to E. coli MG1655 (pSoxS-lux).
As one can see from Fig 5, activation of the PsoxS promoter occurs when BBH is added at all concentrations from 1 to 100 g/l. One-way ANOVA for BBH (all concentrations) and control curves gives p-value < 0.001. On the basis of the data obtained in the experiments we can suggest that BBH causes the appearance of a superoxide anion radical in the cell.

Comparative analysis of biosensors activation
The diagram in Fig 6 demonstrates the activation of biosensors with different BBH concentrations. Activation values were determined as ratio of the area under the curve for each concentration of the test substance to the area under the control curve without the addition of toxicant. Areas (AUC) are obtained by integrating the kinetic curve data shown in graphs 2-5. Statistical analysis was performed by Student's one-sample test.
As can be seen from the diagram in Fig 6, the BBH concentration 100 g/l, having a significant general toxicity for cells, reduces the luminescence of all used biosensors. Only E. coli MG1655 pColD-lux activated by this concentration of BBH exceeds the control curve (after 1.5 hours). This occurs, despite partial cell death, in virtue of the high induction amplitude of the biosensor with the PcolD promoter (up to 20 times). A BBH concentration 10 g/l causes a statistically significant activation of the PcolD, PsoxS, and PoxyR promoters (p < 0.001, one sample t-test), but not the PalkA promoter. The BBH concentration 1 g/l causes a statistically significant activation only of the PsoxS promoter (p = 0.02, one sample t-test). Table 1 shows the threshold concentrations of UDMH and BBH possible to activate stress promoters. As control, data are given for standard, promoter-specific toxicants inducing a

Discussion
It is known that degrading of a number of hydrocarbon compounds leads to reactive oxygen species generation and oxidative stress induction in bacterial cells [25,26].
According to the literature, BBH must be oxidized by oxygen, as well as all hydrocarbons, by a free-radical chain mechanism [27]. In [27] it was shown on the example of cyclopropane  that hydroxyl radical is one of the products of strained compounds destruction under intense light. The BBH molecule includes two elements with strained bonds, which determine a higher energy release in the oxidation process compared to conventional non-strained hydrocarbons.
In [3] comparison of ΔH 6 ¼ (in Kcal/mol) obtained for ring opening of cycloalkanes provided and the ring strain energy is shown to be 3.57 times higher in cyclobutanes compared to cyclopentane. The radical mechanism of oxidation along with the increased reaction energy suggests that the toxic effect of this type of compounds should be determined mainly by reactions of formation of reactive oxygen species. An increased content of superoxide anion radical in the cell can lead to DNA damage [28][29][30]. In this regard, it should be noted that according to our experiment, the toxicity of BBH, determined by the appearance of a superoxide anion radical in cells, is close to that of UDMH, despite the fact that the tests were conducted with an undispersed form of the product insoluble in water, whose contacts with biological objects are sharply reduced and are determined only by the interface of the phases. Under natural conditions, deep dispersion of products is completely absent during fuel spillage, including a humid environment. Thus, the chosen experimental conditions achieve convergence with the conditions of accidental fuel spill. The genotoxic effect of BBH definitely takes place and is expressed by cell damages that activates the following defense systems: SOS response, oxyR/S regulon, and soxR/S regulon.
The time for the appearance of H 2 O 2 in quantities sufficient to activate the cellular response systems after adding BBH to the medium is rather short and is expressed in a minute scale as the PoxyR response to BBH 10 g/l is comparable in speed with the positive control, where 1 mM H 2 O 2 was added at the beginning of incubation. It takes more time for PcolD to respond: 50 minutes-induction onset (T min ), 3.5 hours-maximum response (T max ). This is consistent with the data obtained earlier for other genotoxic substances and is explained by the time spent on entering into the cell, DNA damaging that blocks replication [31], formation of single strand ends and activation of SOS response [10,32]. The long time required for the PsoxS response (T min = 70 min, T max = 4 hours) is apparently explained by the fact that the appearance of superoxide anion radical in the cell is obviously associated only with damage of the respiratory chain [33]. Respiratory chain damage is possible as a result of exposure to alkyl radicals or H 2 O 2 arising from the oxidation of BBH. However, the concentration of H 2 O 2 is significantly less than 1 mM and cannot affect the PsoxS promoter. The permeability of the cell membrane for alkyl radicals arising from BBH oxidation was not investigated before and apparently takes time.
Activation of SOS response occurs only at very high concentrations of BBH in the medium (threshold concentration is about 1 g/l). The test using E. coli MG1655 (pColD-lux) is more sensitive to genotoxic agents than SOS chromotest [34], which is used in toxicology along with the Ames test [35,36] to determine the rate of mutagenesis. These tests correlate well with each other [32], but may underestimate the effect of alkylating compounds on the rate of mutagenesis [37]. In the present work, using the E. coli MG1655 (pAlkA-lux) biosensor, it was shown that during incubation of cells with BBH, DNA alkylation is not observed and, obviously, alkylating compounds do not appear in the medium.
The oxidation of BBH under aerobic conditions in the presence of E. coli cells (Figs 3 and 5, Table 1) leads to the appearance of superoxide anion radical and hydrogen peroxide. As a result, alkyl radicals or alkylhydroperoxides can occur in an organic-rich environment. The addition of catalase to the medium significantly reduces the activation of the oxyR promoter (Fig 3, curve cat-2), which indicates the appearance of hydrogen peroxide, which easily penetrates the cell membrane. Hydrogen peroxide and superoxide radicals, as well as possible alkyl radicals and alkyl hydroperoxides, cause the oxidation of the nitrogenous bases of DNA which accelerates mutagenesis as a result of the inaccurate activity of the DNA polymerase [19,20,[28][29][30].

Conclusion
We believe that the basis of the genotoxic effect of BBH on cells are provided by reactive oxygen species that damage the DNA therefore accelerating mutagenesis. Thus, the mechanism of genotoxic action of BBH is fundamentally different from the action of UDMH, which is determined by alkylating derivatives, primarily nitrosodimethylamine [13] and superoxide-anion radicals arising from the oxidation of UDMH with atmospheric oxygen [12].