Maolida Nihemaiti

Younggun Yoon

Huan He

Michael C Dodd

Jean-Philippe Croué
email: jean.philippe.croue@univ-poitiers.fr

Yunho Lee

Degradation and deactivation of a plasmid-encoded extracellular antibiotic resistance gene during separate and combined exposures to UV254 and radicals

Keywords: Antibiotic resistant bacteria, antibiotic resistance genes, gene transformation, UV, hydroxyl radical, sulfate radical

Introduction

Antibiotic resistance is a natural phenomenon. However, anthropogenic activities (e.g., overuse and disposal of antibiotics) are providing constant selection pressure on antibiotic resistant bacteria (ARB) (Vikesland, Pruden et al. 2017). Antibiotic resistance can be developed and disseminated within the bacterial population by genetic mutation, cell division, and the transfer of antibiotic resistance genes (ARGs) [START_REF] Davies | Origins and Evolution of Antibiotic Resistance[END_REF]. ARGs are contaminants of concern in natural and engineered aquatic systems, as ARG transfer can be linked with the spread of antibiotic resistance to human pathogens [START_REF] Dodd | Potential impacts of disinfection processes on elimination and deactivation of antibiotic resistance genes during water and wastewater treatment[END_REF][START_REF] Pruden | Balancing Water Sustainability and Public Health Goals in the Face of Growing Concerns about Antibiotic Resistance[END_REF].

Mobile genetic elements carrying ARGs (e.g., plasmids, integrons, and transposons) can be disseminated to and expressed by recipient cells through horizontal gene transfer (HGT), including conjugation (mediated by cell to cell contact), transduction (bacteriophage mediated), and transformation (mediated by extracellular DNA) (Lorenz and Wackernagel 1994) Of these, transformation can be understood to represent the most basic mode of HGT, as it does not require the presence of live donor cells or bacteriophages (Lorenz and Wackernagel 1994).

Consequently, quantification of the frequency with which an ARG in extracellular DNA can be transported into and expressed by a recipient cell through transformation (hereafter referred to as transforming activity) can provide a conservative measure of the ability of an ARG to undergo HGT. Transforming activity encompasses (a) efficiency of extracellular DNA transport into a recipient cell, (b) any repair of that DNA by the recipient cell in the event the DNA is damaged, and (c) heritable incorporation and expression of the DNA's genetic information by recipient cells, and is therefore a function of both the recipient cell's ability to import, repair, and express a given ARG and the quality and quantity of the extracellular DNA containing the ARG.

Extracellular DNA can be released into the environment though secretion by live cells and lysis of dead cells. While unstable to nucleases likely to be present in many aquatic matrixes, it can persist in the environment through adsorption onto soil and sediments (Lorenz and Wackernagel 1994, Mao, Luo et al. 2014, Nagler, Insam et al. 2018).

Although conventional disinfection processes during water treatment (e.g., chlorine and UV) efficiently inactivate ARB, intracellular ARGs are more difficult to degrade [START_REF] Mckinney | Ultraviolet disinfection of antibiotic resistant bacteria and their antibiotic resistance genes in water and wastewater[END_REF], Yoon, Chung et al. 2017, He, Zhou et al. 2019) and can be released into water following the death of ARB cells (Zheng, Su et al. 2017). Sub-inhibitory concentrations of disinfectants (e.g., chlorine and monochloramine) were even reported to promote the horizontal transfer of ARGs by increasing the permeability of cell membrane and altering the expression of conjugation-related genes (Guo, Yuan et al. 2015, Zhang, Gu et al. 2017). The proportion of extracellular DNA carrying ARGs was found to increase after various biological and chemical processes in wastewater treatment, revealing the potential risk of antibiotic resistance dissemination in discharged effluent and receiving environments (Liu, Qu et al. 2018, Zhang, Li et al. 2018). Previous studies have reported the enrichment of ARGs in surface water due to the discharged effluents from upstream wastewater plants (Cacace, Fatta-Kassinos et al. 2019, Wu, Su et al. 2019, Osińska, Korzeniewska et al. 2020). High diversity of ARGs in reclaimed wastewater (e.g., agriculture irrigation, potable reuse) may pose risks of antibiotic resistance dissemination to environmental bacteria and even human pathogens (Christou, Agüera et al. 2017, Hong, Julian et al. 2018).

UV-based advanced oxidation processes (UV-AOPs) are increasingly applied for the removal of refractory contaminants during water treatment. The elimination of contaminants in UV-AOP treatment is achieved by dual pathways, namely direct UV photolysis (depending on the UV absorbances and quantum yields of target compounds) and oxidation by powerful radical species [START_REF] Stefan | Advanced Oxidation Processes for Water Treatment[END_REF]. The activation of hydrogen peroxide (UV/H2O2) and peroxydisulfate (UV/S2O8 2-) by UV light produces hydroxyl radical (•OH) and sulfate radical (SO4 •-), respectively, which can degrade a wide range of contaminants, as these two radicals react with many compounds at or near diffusion-controlled reaction rates (Buxton, Greenstock et al. 1988, Neta, Huie et al. 1988).

UV/H2O2 and UV/S2O8 2-have also been reported to efficiently inactivate ARB cells (Michael-Kordatou, Iacovou et al. 2015, Ferro, Guarino et al. 2016, Giannakis, Le et al. 2018).

However, detailed investigation into the reactivity of •OH with intracellular ARGs in these studies was limited due to the significant consumption of •OH by constituents of the water matrix and cell membranes before reaching the cell interiors (Ferro, Guarino et al. 2017, Yoon, Chung et al. 2017, Zhang, Hu et al. 2019). Recent studies have reported that the reactivity of •OH with extracellular chromosomal ARGs is highly dependent on the length of the monitored ARG targets (He, Zhou et al. 2019). More studies are needed to assess whether the observed reactivities are generally applicable to other types of DNA such as plasmid-encoded ARGs. To the best of the authors' knowledge, little is known on the fate of ARGs during SO4 •--based treatment processes.

Quantitative polymerase chain reaction (qPCR) has been widely applied to quantify structurally-intact ARGs or to measure their degradation rates in water and wastewater treatment [START_REF] Mckinney | Ultraviolet disinfection of antibiotic resistant bacteria and their antibiotic resistance genes in water and wastewater[END_REF], Luby, Ibekwe et al. 2016, Hiller, Hübner et al. 2019). It should be noted that qPCR methods usually record the degradation of a small fraction of a target ARG's sequence length (with amplicon sizes typically <1000 bps), and not the entirety of a gene [START_REF] Mckinney | Ultraviolet disinfection of antibiotic resistant bacteria and their antibiotic resistance genes in water and wastewater[END_REF], Chang, Juhrend et al. 2017, Yoon, Dodd et al. 2018). In addition, the efficiency of DNA replication (particularly of damaged DNA) in qPCR methods can be different from that observed in bacterial cells subjected to transformation with the same DNA.

Furthermore, the uptake and repair of DNA by recipient cells can play important roles in modulating gene transforming activity (Chang, Juhrend et al. 2017, Yoon, Dodd et al. 2018).

While the correlation between the degradation of target ARG amplicons (measured by qPCR) and the elimination of ARG transforming activity was reasonably well explained for chromosomal ARGs (He, Zhou et al. 2019), it is still less clear for plasmid-borne ARGs.

Exposure of DNA to UV and •OH is known to induce different types of DNA damage [START_REF] Von Sonntag | Free-radical-induced DNA damage and its repair: A chemical perspective[END_REF]. UV-C generates cyclobutane-pyrimidine dimers (CPDs) as the predominant DNA lesions, while •OH can induce DNA damage ranging from base oxidation to phosphatesugar backbone breakages [START_REF] Görner | New trends in photobiology: Photochemistry of DNA and related biomolecules: Quantum yields and consequences of photoionization[END_REF][START_REF] Sinha | UV-induced DNA damage and repair: a review[END_REF][START_REF] Von Sonntag | Free-radical-induced DNA damage and its repair: A chemical perspective[END_REF]. It is currently not completely clear how the various type(s) of DNA damage resulting from UV, •OH, and SO4 •-are related to the deactivation of ARGs; for example, whether DNA lesions such as CPDs (formed by direct UV irradiation) have the same effect on DNA transforming activity as phosphate-sugar backbone breakage by •OH (generated by H2O2 or S2O8 2-photolysis).

Furthermore, in UV-AOPs, DNA lesions can be generated by both direct UV irradiation and radicals simultaneously.

The aim of this study was to investigate the fate of plasmid-encoded e-ARGs during the common UV at 254 nm water disinfection process (UV254) and its related AOPs. Plasmid (pUC19) encoding an ampicillin resistance gene (amp R ) was purified from Escherichia coli (E. coli) host cells and exposed to UV254, to UV254 in combination with •OH or SO4 •-(by using UV254/H2O2 and UV254/S2O8 2-), or to •OH only (by combining UV at >290 nm with H2O2) (i.e., UV>290/H2O2). UV>290/H2O2 treatment was chosen to study the effect of •OH only exposure because irradiation of H2O2 by UV>290 can generate •OH, while neither UV>290 irradiation nor H2O2 directly affect amp R under these experimental conditions (detailed in Section 3.1). The treated plasmid samples were analysed by qPCR, gel electrophoresis, and ARG transformation assay. DNA repair-proficient E. coli AB1157 (wild-type), as well as repair-deficient E. coli mutants including AB1886 (uvrA -), AB2463 (recA -), AB2480 (uvrA -, recA -), and DH5α (recA -, endA -) were employed as recipient cells in the ARG transformation assay. Competition kinetics methods using radical probe compounds were applied to measure the second-order rate constants of •OH and SO4 •-with amp R segments. Experiments were also conducted in the presence of wastewater effluent organic matter (EfOM) to study the effect of the dissolved organic matter (DOM) on the removal of e-ARGs.

Materials and Methods

Chemical Reagents

All chemicals were in analytical grade or higher and used as received without further purification. Ampicillin sodium salt (#A0166), agar powder (#A1296), sodium chloride (≥99.5%), para-chlorobenzoic acid (99%), and nitrobenzene (≥99%) were purchased from Sigma-Aldrich. Tryptone (#1612) and yeast extract (#1702) were supplied from Laboratorios CONDA. Hydrogen peroxide 30% (Thermo Fisher Scientific) and sodium peroxydisulfate (≥98%, Sigma-Aldrich) were used to prepare the stock solutions of H2O2 and S2O8 2-, respectively. All solutions were prepared in Milli-Q water (18 MΩ⋅cm, Millipore). EfOM was previously extracted from the discharged effluent of a wastewater treatment plant in Jeddah, Saudi-Arabia, with XAD resins (Zheng, Khan et al. 2014). Solutions of EfOM were prepared by dissolving the hydrophobic and transphilic fractions (2:1 by mass) of EfOM in phosphate buffer.

Plasmid Preparation

Plasmid pUC19 was extracted from E. coli DH5α. pUC19 (2686 bp) is a commercially available E. coli vector carrying an ampicillin resistance gene (amp R , 861 bp). One hundred µL of E. coli DH5α mid-exponential growth phase culture in LB broth medium with 50 mg/L of ampicillin was transferred into 150 mL of LB broth medium containing 50 mg/L of ampicillin and incubated overnight (200 rpm, 37°C). Plasmids were extracted from this overnight stock solution using an AccuPrep Plasmid Mini Extraction kit [START_REF] Bioneer | AccuPrep® Nano-Plus Plasmid Mini/Midi/Maxi Extraction Kit[END_REF]. The concentration of recovered plasmid DNA was measured on a NanoDrop ND-2000 Spectrophotometer (NanoDrop Products, Wilmington, USA).

UV Experiments

UV254-based experiments were carried out with a UV quasi-collimated beam device equipped with a low-pressure mercury lamp emitting UV light primarily at 254 nm (Sankyo Denki Ltd., Tokyo, Japan). The UV light was collimated onto the experimental solution, which was contained in a petri dish placed on a magnetic stirrer. The average UV intensity (0.3 mW/cm 2 ) was determined with atrazine chemical actinometry (Lee, Gerrity et al. 2016). Experimental solutions were prepared in autoclaved 2 mM phosphate buffer at pH 7. The initial concentration of plasmid DNA used in experiments was 0.3 or 1 µg/mL. The applied UV fluences ranged from 0-180 mJ/cm 2 . The initial concentrations of H2O2 and S2O8 2-were 0.5 mM for UV254/H2O2 and UV254/S2O8 2-, respectively. UV>290/H2O2 experiments were conducted using a Model 66924 arc lamp source fitted with a 450-W O3-free Xe arc lamp and focusing collimator, and equipped with an atmospheric attenuation filter and dichroic mirror to restrict lamp output to near-UV wavelengths (290 nm < λ < 400 nm) (Newport-Oriel Model 66924; Stratford, CT). Relatively high H2O2 concentrations (i.e., 10 mM) were applied for UV>290/H2O2, in order to increase the yield of •OH due to the low absorbance of UV light by H2O2 in this wavelength region. Samples were withdrawn from the irradiated solutions in each experimental setup at predetermined time intervals for DNA and probe compound analyses. Residual H2O2 and S2O8 2-were quenched with excess bovine catalase and sodium thiosulfate, respectively. Samples for DNA analysis (500 µL) were stored at -20°C and analysed within 24 h. Control experiments indicated that H2O2 and S2O8 2-did not affect qPCR analyses within these experimental conditions (Figure S1).

Radical probe compounds

para-chlorobenzoic acid (pCBA) was used as •OH probe during UV254/H2O2 and UV>290/H2O2 experiments. Both pCBA and nitrobenzene were applied during UV254/S2O8 2-.

Nitrobenzene can be used as a selective •OH probe compound ( • =3.9×10 9 M -1 s -1 ) (Buxton, Greenstock et al. 1988) due to its low reactivity to SO4 •-( • <10 6 M -1 s -1 ) (Neta, Madhavan et al. 1977), whereas pCBA reacts with both radicals (i.e.,

• =3.6×10 8 M -1 s -1 and • =5×10 9 M -1 s -1 ) (Neta, Madhavan et al. 1977, Buxton, Greenstock et al. 1988). pCBA and nitrobenzene were analysed on a high-performance liquid chromatograph (HPLC, Dionex Ultimate 3000, Thermo Scientific) equipped with an XDB-C18 column (5 µm, 4.6 × 150 mm, Agilent). The mobile phase comprised acetonitrile and 10 mM phosphoric acid (40:60, v/v). The two probe compounds were analysed at their maximum absorbance wavelengths (i.e., 240 and 270 nm for pCBA and nitrobenzene, respectively).

qPCR Analysis

Gene degradation was measured by qPCR using a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Four different amplicons (i.e., 192, 400, 603, and µL of autoclaved DNase free water. The qPCR protocol included one cycle at 95°C for 2 min, 30 cycles at 95°C for 5 s, an annealing step at 55°C for 60 s, and an elongation at 72°C for 20 s, followed by a melt curve analysis from 65°C to 95°C. Each sample was analysed in triplicate.

More information on qPCR analysis, the DNA sequence of amp R , and the monitored qPCR target amplicons is described elsewhere (Yoon, Dodd et al. 2018).

Gel Electrophoresis Analysis

pUC19 plasmid (1 µg/mL) treated by UV254 and •OH (UV>290/H2O2) was analysed by gel electrophoresis to investigate the structural change of plasmid DNA. Linearized pUC19 was also prepared for a reference by incubating the plasmid with type II restriction enzyme EcoRI (NEB, USA) at 37 °C for 1 h, followed by enzyme inactivation at 65°C for 20 min. Plasmid samples from UV254, •OH (UV>290/H2O2), and enzyme treatments, as well as a 1 kb DNA ladder (Enzynomics, Korea) were loaded on 0.8% agarose gels at 4 V cm -1 for 35 min. The bands were visualized by ethidium bromide staining. Gel images were captured on a UV transilluminator (Universal mutation detection system, UVP, LLC, USA).

Gene Transformation Assays

Non-ampicillin resistant E.coli K12 bacteria strains DH5α (recA -, endA -), AB1157 (wildtype), AB1886 (uvrA -), AB2463 (recA -), and AB2480 (uvrA -, recA -) were used as recipient cells for gene transformation assays. DH5α strain was commercially available from ATCC;

others were provided by CGSC (Coli Genetic Stock Center at Yale University). Details on transformation assays were described by Yoon, Dodd et al. (2018). In brief, competent cells were prepared by chemical treatment of non-resistant E. coli K12 strains using calcium chloride and glycerol (Shanehbandi, Saei et al. 2013) and preserved at -80°C until use. Fifty µL of treated plasmid sample was mixed with 100 µL of thawed competent cells. After incubating in ice for 30 min, the mixture was quickly transferred onto a digital test tube heater (45°C) for 45 s and then placed back in ice for 2 min. After the heat shock, the samples were mixed with 900 µL of LB broth and cultured for 45 min (200 rpm, 37°C). Finally, the incubated samples were serially diluted with LB broth and plated onto two types of LB agar plates (i.e., with and without inclusion of 50 mg/L of ampicillin). After 24 h of incubation at 37°C, the number of ARB colonies (transformants) detected on selective agar plates (with ampicillin) were compared with the total recipient cells growing on nonselective agar plates (without ampicillin). The gene transformation efficiency was calculated as follows:

Transformation efficiency = !"" #$%$&'()$ *%+'$ (-.// 1) " ! 343! !"" 565#$%$&'()$ *%+'$ (-.// 1)
The typical concentrations of total recipient cells, as measured by culturing on nonselective agar plates (without ampicillin), were approximately 3×10 8 and 2×10 8 CFU/mL for E. coli DH5α and E. coli AB strains, respectively. Control experiments conducted by directly plating the recipient cells (without heat shock) on nonselective plates showed <10% variation in the final colony counts of recipient cells as compared to the conditions incorporating heat shock (Figure S3), and <50% variation in colony counts between strains. These results indicated that the type of bacterial strain used, or stresses to which the strains were subjected during transformation assays (e.g., heat shock), did not significantly impact the fitness or growth rates of recipient cells on nonselective plates. Calibration curves prepared with known concentrations of plasmid DNA indicated that the number of transformants linearly increased with increasing plasmid DNA concentration from 10 -5 -10 -1 µg/mL (Figure S4). The transformation efficiency of amp R calculated for E. coli K12 strains was in the range of 10 -4 -10 -8 , which was comparable with previous studies (Chang, Juhrend et al. 2017, Yoon, Dodd et al. 2018).

Statistical analysis

Statistical analysis was conducted using SigmaPlot 12.0 and GraphPad Prism 7.0. The fluence-based rate constants for gene degradation and transformation efficiency loss under various experimental conditions were compared by multiple linear regression analysis. The null hypothesis was that the first-order rate constants were identical, with p=0.05 as the threshold significance level. UV254 exposure (Figure 1a). The solution containing pUC19 was irradiated with UV254 light (0-130 mJ/cm 2 ). The degradation of amp R segments (192, 400, 603, and 851 bp) measured by qPCR followed first-order kinetics with respect to UV fluence (r 2 >0.99) (Figure 1a). The 192 bp amp R segment was degraded by about 1-log at 110 mJ/cm 2 , whereas 4-log reduction was already achieved for the 851 bp segment, which was close to the overall size of amp R (861 bp). ), based on an assumption that the UV reactivity per base pair is the same across the entire plasmid. The 78,*%+#;(< values estimated from the 192, 400, 603, and 851 bp amplicons were 2.9(±0.06)×10 -1 , 2.3(±0.04)×10 -1 , 2.6(±0.05)×10 -1 , and 2.7(±0.06)×10 -1 cm 2 /mJ, respectively, yielding an average 78,*%+#;(< value of 2.6(±0.05)×10 -1 cm 2 /mJ (Table 1).

Results and Discussions

A theoretical approach was recently employed to estimate the photoreactivity of DNA (i.e., formation rate of CPDs and 6,4-photoproducts) based on the molar absorption coefficients, quantum yields, and the number of 5'-bipyrimidine-3' doublets of a given DNA (Yoon, Dodd et al. 2018, He, Zhou et al. 2019). By applying this approach, the overall DNA lesion formation rate on pUC19 upon UV254 exposure, +%% %$#(65# , was calculated as 2.1×10 -1 cm 2 /mJ (Text S1), which was close to the average 78,*%+#;(< (2.6×10 -1 cm 2 /mJ) predicted from qPCR results as mentioned above. Experimental conditions: pUC19: 1 µg/mL, 2 mM phosphate buffer at pH 7, fluence range for UV254: 0-130 mJ/cm 2 , 10 mM of H2O2 and 1 µM of pCBA were added in UV>290/H2O2. The apparent rate constant values were reported in the form of Mean ± Standard Error. b The *%+#;(< values of pUC19 plasmid were estimated based on the :;* of amp R segments obtained from qPCR analyses (see Section 3.1 for details).

•OH exposure (Figure 1b). Control experiments have confirmed that amp R segments were stable under UV>290 irradiation alone (Figure S5), which was attributed to the low UV absorbance of plasmid DNA above 290 nm. Moreover, 10 mM (340 mg/L) of H2O2 had negligible effect on amp R segment (Figure S1). Thus, UV>290/H2O2 was applied to investigate the influence of •OH on the degradation of e-ARG. The •OH exposure, E[• GH]J , in the UV>290/H2O2 system was obtained by following the degradation of pCBA, which was additionally added in the experimental solution. No UV>290 direct photolysis of pCBA was observed in the absence of H2O2 (Figure S6). Thus, the •OH exposure was calculated from

E[˙GH] J = - %5 [MNOP] [MNOP] Q R ST,MNOP
• [START_REF] Elovitz | Hydroxyl Radical/Ozone Ratios During Ozonation Processes. I. The Rct Concept[END_REF], where the UV,*WX:

• is the secondorder rate constant of •OH with pCBA (5×10 9 M -1 s -1 ) (Buxton, Greenstock et al. 1988). The logarithmic-scale degradation of amp R segments exhibited linear correlations with •OH exposure during UV>290/H2O2 (Figure 1b). The resulting bimolecular rate constants derived from the slopes of linear curves, UV,:;* • = 2.303 × slope, are summarized in Table 1.

The UV,:;* • of amp R segments increased with amplicon size, ranging from 8.1×10 9 M -1 s -1 for the 192 bp amplicon to 5.6×10 10 M -1 s -1 for the 851 bp amplicon (Table 1). A good linear relationship (r 2 =0.996) was observed between the UV,:;* • and the base pair number of amp R segments (Figure S7), which was consistent with previous findings that •OH non-selectively reacts with all nucleotides in a DNA sequence (both AT and GC contents) (He, Zhou et al. 2019). The UV,*%+#;(< • of the entire plasmid was predicted to be 1.8×10 11 M -1 s -1 by applying the base pair number of pUC19 (2686 bps) into the regression equation of this linear curve (Figure S7). Alternatively, similar to the 78,*%+#;(< as mentioned above, the UV,*%+#;(< • of which is smaller than its linear form and migrates faster in electrophoresis gel. The UV254 irradiation (0-130 mJ/cm 2 ) did not change the position of the band, suggesting that the pUC19 kept its supercoiled form without significant conformational change (e.g., strand breakage). It is well documented that the gene damage by UV254 is mainly caused by the formation of DNA lesions such as CPDs and 6,4-photoproducts (pyrimidine adducts) [START_REF] Sinha | UV-induced DNA damage and repair: a review[END_REF]Häder 2002, Cadet and[START_REF] Cadet | Formation of UV-induced DNA damage contributing to skin cancer development[END_REF], which are generally not detectable by gel electrophoresis analysis (Yoon, Dodd et al. 2018).

The reaction pathways of •OH with DNA include addition to nucleobases -producing adduct radicals -and hydrogen-abstraction from 2-deoxyribose (sugar moiety) (von Sonntag 2006, Dizdaroglu and Jaruga 2012), resulting in not only oxidized bases and abasic sites, but also strand breaks (Cadet, Delatour et al. 1999[START_REF] Von Sonntag | Free-radical-induced DNA damage and its repair: A chemical perspective[END_REF]. The double-strand breaks are expected to be mainly responsible for the conformational change from supercoiled to linear plasmid form. Gel electrophoresis analysis showed that the pUC19 plasmid band gradually moved from the supercoiled to linear form position with increasing •OH exposure (Figure 2b), revealing the formation of double-strand breaks in pUC19. Bacterial cells have developed the ability of repairing DNA lesions induced by UV irradiation and oxidant stress (e.g., radicals) using several enzyme systems (Teebor, Boorstein et al. 1988, Sinha and[START_REF] Sinha | UV-induced DNA damage and repair: a review[END_REF]. The biological deactivation of pUC19 upon separate treatment with UV254 and •OH was studied by analysing its transformation of non-resistant recipient cells having different DNA repair abilities. The decrease of transforming activity of pUC19 followed first-order kinetics with respect to UV fluence or •OH exposure, and was variable depending on the recipient cells used (Figure 3). The most rapid decrease was observed for AB2480 (uvrA -, recA -) during each separate treatment with UV254 and •OH. No transformant was detectable from AB2480 (more than 4-log decrease) when the UV fluence was above 5 mJ/cm 2 , or when the •OH exposure was higher than 5.5×10 -11 M•s. Thus, the fluence-based rate constant for UV ( 78,'C+5#D6C;+'( 65) and second-order rate constant for •OH ( UV,'C+5#D6C;+'( 65• ) for the transforming activity loss for AB2480 were not calculated in this case due to the limited data points.

During UV254 exposure (Figure 3a), the loss of transforming activity was slower in AB1886 (uvrA -, 0.24 cm 2 /mJ) than AB2480, followed by AB2463 (recA -, 0.16 cm 2 /mJ), AB1157 (wildtype, 0.10 cm 2 /mJ), and DH5α (recA -, endA -, 0.07 cm 2 /mJ), in which the values in parentheses show 78,'C+5#D6C;+'(65 (Table 1). The variation in the transformation efficiency of plasmids carrying the same DNA damage indicated that the UV-induced damage can be repaired by recipient cells to different extents, depending on their repair gene proficiencies (or deficiencies).

The uvrABC proteins involve the nucleotide excision repair of a variety of DNA lesions including the UV-induced CPDs and 6,4-photoproducts. This process is initiated by the interaction of uvrA protein with DNA to recognize the damaged sites (Kisker, Kuper et al. 2013). The recA protein is essential for the post-replication repair of double-strand breaks and single-strand gaps (e.g., T-T dimers) by homologous recombination [START_REF] Smith | recA-dependent DNA repair processes[END_REF]Wang 1989, Shinohara and[START_REF] Shinohara | Homologous recombination and the roles of double-strand breaks[END_REF]. These roles of uvrA and recA proteins well explained the trend observed from the transformation rates of E. coli strains. AB2480 was most sensitive to UVinduced DNA damage on pUC19 due to lack of both uvrA and recA genes. The uvrA deficient AB1886 was the second most sensitive, followed by the recA deficient AB2463, indicating that uvrA played a more important role than recA for the repair of UV-induced DNA damage. The wild-type AB1157 was less sensitive than the other three E. coli mutant strains owing to its DNA repair capability by uvrA and recA. Interestingly, despite its deficiency in recA, the DH5α strain exhibited even lower sensitivity to UV-induced DNA damage compared to wild-type AB1157. This was possibly attributable to the lack of a functional endA gene in DH5α. The endA gene is associated with the formation of extracellular DNase I, which can hydrolyze exogenous plasmid DNA, consequently reducing its uptake and transformation (Shou, Kang et al. 2019). In line with this, the DH5α showed ∼10-fold higher transformation efficiency than the other E. coli strains proficient in endA when all were transformed with equivalent concentrations of undamaged pUC19 (Figure S4). This endA deficiency might therefore render E. coli DH5α inherently more active for DNA damage repair than the other strains, despite its lack of recA. 3b), the rate of transforming activity loss was the highest for AB2480 followed by AB2463 (recA -, 8.2×10 10 M -1 s -1 ), AB1886 (uvrA -, 6.5×10 10 M -1 s -1 ), AB1157 (wild-type, 4.1×10 10 M -1 s -1 ), and DH5α (recA -, endA -, 2.9×10 10 M -1 s -1 ) in which the values in parentheses show UV,'C+5#D6C;+'( 65• (Table 1). The recA deficient AB2463 showed higher sensitivity to •OH-induced DNA damage than the uvrA deficient AB1886, which was opposite compared to the case for UV254 (Figure 3a). This suggests that recA is more efficient for repairing •OH-induced DNA damage, as homologous recombination is based on exchange of (damaged) nucleotide sequences (Alberts, Johnson et al. 2002) and capable of repairing not only base oxidation but also double-strand breaks. In contrast, uvrA is based on nucleotide excision repair and more specific to the repair of UV-induced DNA lesions (base modification such as CPDs), but not efficient for double-strand breaks (Alberts, Johnson et al. 2002). (UV>290/H2O2). Specific kinetic parameters are summarized in Table 1 and error bars represent the standard errors of apparent rate constants. Rate constants for the loss of transforming activity in AB2480 were not calculated due to the limited data points.

During •OH exposure (Figure
The apparent rate constants for the degradation of amp R segments, pUC19 plasmid, and the loss of gene transforming activity are compared in Figure 4. For both UV254 direct photolysis and •OH oxidation, the estimated degradation rates of the entire plasmid (based on qPCR analyses) overestimated the rates of pUC19 deactivation (loss of transforming activity) observed for all repair-proficient E. coli recipient strains, which can be explained by the repair of UV254and •OH-induced damage by repair-proficient recipient cells. Our results were in agreement with previous findings on the UV254 treated pWH1266 plasmid and its transformation of Acinetobacter baylyi (Chang, Juhrend et al. 2017). However, the rate of gene deactivation during UV254 direct photolysis measured using uvrA deficient AB1886 as the recipient strain was close to the estimated degradation rate of pUC19 and the formation rate of DNA lesions (Figure 4a,b). The minimum rates of gene deactivation measured by double mutant AB2480 (uvrA -, recA -) were estimated based on the limited data points shown in Figure 3, which gave 0.91 cm 2 /mJ and 1.7×10 11 M -1 s -1 for UV254 and •OH exposure, respectively. These values were significantly larger than the gene deactivation rates measured by any of the repair proficient strains. Furthermore, the degradation rate of the entire plasmid underestimated the loss of pUC19 transforming activity measured by AB2480 (uvrA -, recA -) during UV254 direct photolysis and •OH oxidation. The degradation of amp R and the elimination of gene transforming activity (E. coli DH5α as recipient cells) were tested during combined exposure to UV254 and radicals (•OH and SO4 •-), in comparison to UV254 direct photolysis. The degradation of amp R amplicons followed first-order kinetics with respect to UV fluence (r 2 >0.99) during UV254, UV254/H2O2, and UV254/S2O8 2- treatments (Figure 5). A lower initial concentration of pUC19 (0.3 µg/mL) and a higher fluence range (0-180 mJ/cm 2 ) were applied in these tests than in the experiments described in the preceding sections. However, the degradation rate constants of amp R amplicons upon UV254 direct photolysis (i.e., 2.4×10 -2 -8.2×10 -2 cm 2 /mJ) (Table S1) were in good agreement with those obtained during the treatment of 1 µg/mL of pUC19 by 0-130 mJ/cm 2 of UV254 (i.e., 2.0×10 -2 -8.9×10 -2 cm 2 /mJ, Table 1). The 78 YZ /V Y U Y of amp R amplicons during UV254/H2O2 were in the range of 1.0×10 -1 -3.0×10 -1 cm 2 /mJ, which were larger than the 78 YZ by a factor of ∼4 (Table S1). Therefore, the contribution of UV254 direct photolysis to the overall gene degradation during UV254/H2O2 was only about 25% ( 78 YZ / 78 YZ /V Y U Y × 100%), revealing the significant role of •OH exposure (75%).

e-ARG degradation and deactivation by combined exposure to UV254 and radicals

The 78 YZ /V Y U Y for the elimination of gene transforming activity was (5.1±0.2)×10 -2 cm 2 /mJ, which was larger than the 78 YZ by a factor of only 1.1 ( 78 YZ /V Y U Y / 78 YZ = 1.1, Table S1).

Thus, even though the •OH exposure significantly accelerated gene degradation (by a factor of 4), its contribution to the elimination of gene transforming activity was not as efficient as expected. Figure 6 summarizes the results from this study and the literature on the effect of •OH exposure on degradation of the 851 bp amp R segment and loss of gene transforming activity (measured using E. coli DH5α as recipient strain). Plasmid pUC4k or pUC19 carrying amp R was treated with UV/H2O2 at different conditions of UV wavelength and initial H2O2 concentrations, i.e., UV254/H2O2 (0.3 mM or 0.5 mM) and UV>290/H2O2 (10 mM). The •OHinduced degradation or deactivation were calculated by subtracting the effect of UV254 direct photolysis from the overall results of corresponding UV254/H2O2. Although the statistical analysis showed a significant variation (p=0.0149) in the slopes of linear curves in Figure 6a, the UV,B`= @* • values during UV254/H2O2 treatment differed only by factors of 0.8-1.5 from that during •OH only process (UV>290/H2O2). This suggested that UV254-induced gene damage on the plasmid (e.g., CPDs and pyrimidine adducts) did not cause significant impact on the reactivity of •OH with nucleobase and sugar moieties during UV254/H2O2. However, the elimination of gene transforming activity (measured using E. coli DH5α as recipient strain) varied significantly in the different treatments. The loss of gene transforming activity was ~5 times slower in UV254/H2O2 processes (UV254 and •OH co-exist) as compared to that obtained from UV>290/H2O2 (•OH only). This indicates that the elimination of gene transforming activity caused by UV254 direct photolysis and •OH oxidation were not additive during UV254/H2O2. One possible explanation could be that some DNA damage repair pathways such as nucleotide excision repair by uvrABC also repaired •OH-induced lesions (e.g., base oxidation) located near the UV-induced lesions, leading to much less extensive loss of transforming activity than would be expected from simple summation of the losses in transforming activity observed for UV254 and •OH exposure separately.

The degradation rates of amp R amplicons (1.0×10 -1 -3.2×10 -1 cm 2 /mJ) during UV254/ S2O8 2- were also ~4-fold larger than that obtained during UV254 (Table S1). SO4 •-is the primary radical generated during UV254 photolysis of S2O8 2-. However, SO4 •-can further react with H2O, OH -, and Cl -(sometimes present as an impurity) in aqueous solution to produce •OH (Lutze, Kerlin et al. 2015). Using the degradation kinetics of radical probe compounds (nitrobenzene and pCBA) (Figures S7 andS8), the steady-state concentration of •OH during UV254/S2O8 2-was determined to be 6.4×10 -13 M, which was approximately two orders of magnitude smaller than that of SO4 •-(1.8×10 -11 M) under experimental conditions used here (Text S3). This was consistent with the theoretically estimated values considering all the possible reactions related to

•OH formation in UV254/S2O8 2-system (Text S4). Despite the much lower concentration, the contribution of •OH to the overall gene damage cannot be ignored, as the amp R segments appeared to be much more reactive to •OH than SO4 •-(see discussions below). Thus, the percentages of gene degradation attributable to UV254 direct photolysis, SO4 •-, and •OH during UV254/S2O8 2-treatment could be estimated as ~23%, ~33%, and ~44%, respectively, in which the relative contributions of each pathway to the overall gene damage were calculated based on the observed rate constants for degradation of amp R amplicons during UV254 and UV254/S2O8 2-treatments, the steady-state concentrations of •OH and SO4 •-in UV254/S2O8 2- experiments, and the second-order rate constants for reactions of • OH and SO4 •-with the amp R amplicons (Text S5).

The second-order rate constants of SO4 •-with amp R segments were determined to be in the range of (1.07-1.96)×10 9 M -1 s -1 (Text S5 and Table S2), which were about an order of magnitude lower than the •OH rate constants (Table 1). SO4 •-preferentially adds to the electronrich sites of the nucleobases (e.g., C5 of pyrimidines) to generate radical adducts, which decay to nucleobase radicals as intermediates, followed by hydroxylation and oxidation in aqueous solution [START_REF] Von Sonntag | Free-radical-induced DNA damage and its repair: A chemical perspective[END_REF]. However, SO4 •-is larger than •OH and negatively charged.

Therefore, steric hindrance and electrostatic repulsion might decrease the reactivity of SO4 Similar to UV254/H2O2 processes, the elimination rate of pUC19 transforming activity during UV254/S2O8 2-treatment was also only slightly larger than that in UV254 direct photolysis S1), even while the observed amplicon degradation rates were ~4-fold faster for UV254/S2O8 2-compared to UV254 direct photolysis. This further supports the above findings that co-exposure to UV254 and radicals during UV254-AOPs did not lead to faster transforming activity loss than the separate exposures to UV254 and radicals, due to the facile repair of UV254 and/or radical-induced DNA damage in host cells. The efficiencies of amp R degradation during UV254, UV254/H2O2, and UV254/S2O8 2- experiments were investigated in the presence of EfOM (5.4 mg C/L). The UV 254 nm absorbance of EfOM solution (a254) was 0.1 cm -1 . The path length in the petri dish (L) was 1.6 cm. Thus, the water factor, WF=(1-10 -a254⋅L )/(a254⋅L⋅ln10) [START_REF] Bolton | Standardization of methods for fluence (UV Dose) determination in bench-scale UV experiments[END_REF], was calculated to be 0.84. The average UV intensity in the solution containing EfOM was then corrected from 0.30 mW/cm 2 to 0.25 mW/cm 2 using the calculated water factor (Lee, Gerrity et al. 2016). The amp R segments were gradually degraded with increasing UV fluence following first-order kinetics (Figure 7). Interestingly, the 78,:;* of each segment during UV254 irradiation in the presence of EfOM was approximately 1.2 times higher than the corresponding conditions without EfOM (p<0.0001) (Table S1). Chromophoric dissolved organic matter (DOM) is known to generate reactive species upon UV irradiation, such as triplet excited states of DOM ( 3 DOM*), singlet oxygen, and •OH, which have been proven to contribute to the oxidation of organic pollutants (Chin, Miller et al. 2004, Lester, Sharpless et al. 2013, Batista, Teixeira et al. 2016[START_REF] Rosario-Ortiz | Probe Compounds to Assess the Photochemical Activity of Dissolved Organic Matter[END_REF]. Bacteriophage MS2 was found to be inactivated by 3 DOM* and singlet oxygen generated from sunlight irradiation of wastewater and river water DOM isolates (Kohn, Grandbois et al. 2007[START_REF] Kohn | Sunlight-Mediated Inactivation of MS2 Coliphage via Exogenous Singlet Oxygen Produced by Sensitizers in Natural Waters[END_REF], Rosado-Lausell, Wang et al. 2013). The reactive oxygen species (e.g., singlet oxygen, superoxide, H2O2) generated during photooxidations involving 3 DOM* were proposed to inactivate bacteria by damaging DNA, proteins, and cell membranes (Song, Mohseni et al. 2016, Nelson, Boehm et al. 2018). Our findings suggest that through its photosensitizing properties, DOM can indirectly contribute to the photodegradation of e-ARGs. Similar results were reported recently on the enhanced photo-degradation of pBR322 plasmid-encoded tet A and blaTEM-1 genes in DOM solution (Zhang, Li et al. 2019). This degradation pathway might play an especially important role during solar disinfection processes, due to the low UV absorbance characteristics of DNA at high wavelengths (and consequent minimal direct UV photolysis under such conditions).

( 78 YZ /a Y U b Y / 78 YZ = 1.1, Table
In contrast, EfOM mainly acted as radical scavenger during UV254/H2O2 and UV254/S2O8 2- experiments. The 78 YZ /V Y U Y and 78 YZ /a Y U b Y of amp R segments in the presence of EfOM were each decreased by a factor of ∼3 compared to the conditions without EfOM (Table S1). The degradation kinetics of pCBA and nitrobenzene (Figure S10) also confirmed the much lower levels of radicals in the presence of EfOM (e.g., [SO4 •-]ss: 9.5×10 -13 M with EfOM vs. 1.8×10 -11 M without EfOM). Overall, the degradation efficiency of amp R in the presence of EfOM

Conclusions

• The degradation of amp R segments and the loss of gene transforming activity followed firstorder kinetics with respect to UV fluence and •OH exposure during UV254 direct photolysis and •OH oxidation (UV>290/H2O2). The degradation rate constants of amp R segments increased with amplicon size, which corresponded to the increase in nucleotide bps (and hence, reaction sites) with increased amplicon length. Gel electrophoresis results indicated that UV254 (0-130 mJ/cm 2 ) did not change the supercoiled conformation of pUC19 plasmid, whereas •OH exposure (0-2.7×10 -10 M⋅s) led to strand-breaks.

• The loss of gene transforming activity varied depending on the type of recipient cells. Double mutant AB2480, which was deficient in repair genes uvrA and recA, was the most sensitive to DNA damage induced by UV254 direct photolysis and •OH oxidation. The predicted degradation rates for the entire pUC19 plasmid overestimated the loss in transforming activity of pUC19 for all recipient cells other than AB2480, apparently due to the repair of DNA damage by repair-proficient strains.

• The second-order rate constants of •OH with amp R segments were determined to be in the range of 8.1×10 9 -5.6×10 10 (M -1 s -1 ) and showed a strong linear relationship with the base pair number of amp R segments, suggesting that •OH reacts with all nucleobases of plasmid DNA non-selectively. The rate constants of SO4 •-with amp R segments were an order of magnitude lower than the corresponding •OH rate constants; thus, the contribution of the trace amount of •OH to the overall gene damage cannot be ignored during UV254/S2O8 2- treatment.

• Although •OH and SO4 •-significantly accelerated (~75%) the degradation of amp R amplicons measured by qPCR during UV254/H2O2 and UV254/S2O8 2-experiments, this only marginally increased the gene deactivation rate (~11%), as gene damage caused by UV254 and radical co-exposure appeared to be repaired more efficiently by recipient cells than the gene damage from the separate UV254 and radical exposures. Thus, the elimination efficiency of the ARGs' transforming activities was similar for UV254 vs UV254-AOPs at the same UV fluence, and the extent of elimination was mainly determined by the level of UV fluence. Because the transforming activity of the amp R gene could be lowered by only ∼1log at a typical UV disinfection fluence for water treatment (e.g., 40 mJ/cm 2 ) but increased to more than 4-logs at an elevated UV fluence for UV-AOPs (e.g., 500 mJ/cm 2 ), the use of UV-AOPs may still prove beneficial for ARG degradation and deactivation, with the accompanying benefit of improved degradation of trace organic contaminants.

• Reactive species formed from the excitation of chromophoric organic matter appear to contribute to accelerated degradation of amp R during UV254 irradiation in the presence of EfOM (compared to direct photolysis by UV254 alone), whereas EfOM mainly acted as a radical scavenger in UV254/H2O2 and UV254/S2O8 2-experiments.

  851 bp) were monitored, which covered varying fractions of amp R as shown in Figure S2. The longest size of amp R segment targeted in qPCR was 851 bp instead of the whole amp R gene (861 bp), due to the low amplification efficiency of the 861 bp amplicon in the qPCR method. The reaction mixture (20 µL) consisted of 1 µL of each primer, 1 µL of sample, 10 µL of EvaGreen® supermix, and 7

  Figure 1. Degradation of amp R segments during the treatment of pUC19 (1 µg/mL) with (a)

Figure 2 .

 2 Figure 2. DNA electrophoresis gel of pUC19 treated by (a) UV254 and (b) •OH (UV>290/H2O2).

Figure 3 .

 3 Figure 3. Elimination of gene transforming activity during the treatment of pUC19 (1 µg/mL)

Figure 4 .

 4 Figure 4. Apparent rate constants for the degradation of amp R segments, pUC19 plasmid, and

Figure 5 .

 5 Figure 5. Degradation of amp R segments and elimination of gene transforming activity (E.coli DH5α as recipient cells) with increasing UV fluence (0-180 mJ/cm 2 ) during treatment of pUC19 (0.3 µg/mL) with (a) UV254, (b) UV254/H2O2 (0.5 mM), and (c) UV254/S2O8 2-(0.5 mM)

Figure 6 .

 6 Figure 6. (a) Degradation of amp R segment (851 bp) and (b) elimination of gene transforming

3. 5 Figure 7 .

 57 Figure 7. Degradation of amp R segments in the presence of wastewater effluent organic matter

  the entire plasmid (e.g., 78,*%+#;(< = 78,=>?@* ×

	?ABA @*
	=>? @*

The resulting fluence-based rate constants derived from the slopes of linear curves, 78,:;* = 2.303 × slope, are summarized in Table

1

. The 78,:;* of amp R segments increased with amplicon size, which was consistent with the increasing number of potential reaction sites with increasing number of nucleotide bps. The 78,:;* values obtained from this study were comparable to previously-reported values for the same amp R segments

(Yoon, Dodd et al. 2018)

. The 78,*%+#;(< of pUC19 plasmid could be estimated by extrapolating the 78,:;* of each segment to

Table 1 .

 1 Apparent rate constants for the degradation of amp R segments and pUC19 plasmid, and the elimination of gene transforming activity during UV254 and •OH only treatments a

	amp R segments	pUC19 plasmid b	Loss of gene transformation
	#base	:;*	*%+#;(<	average	Recipient	'C+5#D6C;+'(65
	pairs				cells
	(bps)
			UV254 (cm 2 /mJ)
	192	2.0(±0.05)×10 -2 2.9(±0.06)×10 -1		DH5α	6.5(±0.21)×10 -2
	400 603	3.4(±0.07)×10 -2 2.3(±0.04)×10 -1 5.8(±0.11)×10 -2 2.6(±0.05)×10 -1	2.6(±0.05)×10 -1	AB1157 AB1886	1.0(±0.03)×10 -1 2.4(±0.06)×10 -1
	851	8.9(±0.16)×10 -2 2.7(±0.06)×10 -1		AB2463	1.6(±0.06)×10 -1
			•OH (UV>290/H2O2) (M -1 s -1 )
	192	8.1(±0.69)×10 9	1.2(±0.10)×10 11		DH5α	2.7(±0.05)×10 10
	400 603	2.2(±0.07)×10 10 1.5(±0.05)×10 11 3.5(±0.10)×10 10 1.6(±0.04)×10 11	1.5(±0.07)×10 11	AB1157 AB1886	4.1(±0.14)×10 10 6.5(±0.18)×10 10
	851	5.6(±0.27)×10 10 1.8(±0.08)×10 11		AB2463	8.2(±0.23)×10 10
	a

  •- toward negatively charged DNA molecules in comparison to •OH.

Acknowledgements

This study was supported by the Korea Institute of Marine Science & Technology Promotion funded by the National Research Foundation funded by the Ministry of Science, ICT and Future Planning (NRF-2020R1A2C2011951). Curtin University (Curtin International Postgraduate Research Scholarship) and Water Research Australia (WaterRA Postgraduate Scholarship) are gratefully acknowledged for providing financial support for M. Nihemaiti. Additional support for H. He and M. C. Dodd from U.S. National Science Foundation Grant Number CBET-1254929 is gratefully acknowledged.

Abbreviations amp R

pUC19 was also estimated by normalizing the UV,:;* • of each segment (qPCR analyses) to the entire plasmid and calculating the average value as 1.5(±0.07)×10 11 M -1 s -1 (Table 1).

The UV,:;* • values for the plasmid-encoded amp R segments in this study were lower by a factor of ∼3 than those reported for the chromosome-encoded blt genes with similar amplicon length (He, Zhou et al. 2019). To see whether the reactivity difference is due to the conformational difference between the supercoiled-form plasmid and linear-form genomic DNA, the theoretical diffusion-controlled rate constant for the reaction of •OH with supercoiled pUC19 was calculated based on a method proposed by He, Zhou et al. (2019). The theoretical maximum UV

• value was predicted as 3.84×10 11 M -1 s -1 for pUC19, which yields a bp-specific UV • value of 1.43×10 8 MAT+GC -1 s -1 when normalized to the length (in AT+GC bps) of pUC19 (2686 bps) (Text S2). For comparison, the theoretical maximum UV • value was 7.97×10 12 M -1 s -1 for a hypothetical 50-kbp linear-form segment of genomic DNA, yielding a bp-specific UV • value of 1.59×10 8 MAT+GC -1 s -1 (He, Zhou et al. 2019). This indicates that the conformational difference between the plasmid (supercoiled) and genomic (linear) DNA (the bp-specific UV (1.43×10 8 MAT+GC -1 s -1 ) with the length of each amplicon, and ranged from 2.57×10 10 M -1 s -1 for 192 bp to 1.14×10 11 M -1 s -1 for 851 bp (Text S2). Thus, the experimental UV,:;* • for pUC19 were lower by a factor of 2.5 than the theoretical maximum UV,:;* • values. This difference is acceptable considering all possible experimental errors as well as the hypothesis applied in the theoretical calculation.

Structural degradation of plasmid (Figure 2). Figure 2a shows the electrophoresis gel of pUC19 exposed to UV254. The intact pUC19 before the UV254 exposure showed a band located lower than the linearized pUC19 by EcoRI. Plasmid DNA generally exists in a supercoiled form, increased only marginally (within a factor of 1.4) during UV254/H2O2 and UV254/S2O8 2-, 586 compared to UV254 alone, due to the significant scavenging of radicals by EfOM.


D Grillault Laroche

E Curis

F Bellivier

C Nepost

Cindie Courtin

B Etain

Cynthia Marie-Claire

D Grillault Laroche
email: diane.grillaultlaroche@aphp.fr

Al

Childhood maltreatment and HPA axis gene expression in bipolar disorders: A gene network analysis

Keywords: bipolar disorder, childhood maltreatment, stress, gene expression, HPA axis, NR3C1, DGKH

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Bipolar disorder (BD) is a chronic and burdensome psychiatric condition that results from interactions between genetic and environmental risk factors. In this context, childhood maltreatment (CM) has been suggested as a major risk factor for developing BD [START_REF] Aas | The Role of Childhood Trauma in Bipolar Disorders[END_REF]. All subtypes of CM (neglect or abuse) were more frequent and severe in patients with BD as compared to healthy controls, with a predominant role of emotional abuse (EA) (Palmier-Clauss 2016). Two thirds of patients with BD reported multiple types of CM [START_REF] Etain | Preferential Association between Childhood Emotional Abuse and Bipolar Disorder[END_REF].

CM has been suggested to modify the functioning of multiple biological pathways (hypothalamic-pituitary-adrenal (HPA) axis, neurotransmission, immuno-inflammation, or neuroplasticity), but the associated biological disturbances remain poorly described. A metaanalysis of 41 studies focusing on the HPA axis alterations reported an association between BD and significantly increased levels of cortisol and HPA axis dysregulation. This has been suggested to be related to the exposure to environmental risk factors such as CM [START_REF] Murri | The HPA Axis in Bipolar Disorder: Systematic Review and Meta-Analysis[END_REF]). Nevertheless, findings on the association between CM and HPA axis disturbances remain relatively conflicting since the exposure to CM has been associated to both exaggerated and attenuated HPA axis activity. This heterogeneity might be explained by differences in the severity of CM and their unique or repetitive nature [START_REF] Tyrka | Childhood Adversity and Epigenetic Regulation of Glucocorticoid Signaling Genes: Associations in Children and Adults[END_REF]).

Among genes involved in the HPA axis, NR3C1, encoding for the glucocorticoid receptor (GR), has been the most widely studied in association with CM. Indeed, CM has been associated with a lower expression of NR3C1 in leukocytes [START_REF] Bustamante | Glucocorticoid Receptor DNA Methylation, Childhood Maltreatment and Major Depression[END_REF], these results being consistent with a decreased mRNA levels of NR3C1 in the hippocampus of suicide victims who were exposed to childhood abuse [START_REF] Mcgowan | Epigenetic Regulation of the Glucocorticoid Receptor in Human Brain Associates with Childhood Abuse[END_REF]. Furthermore, some studies have suggested that the level of transcripts of NR3C1 were modified in patients with BD as compared with controls, however without further investigations of the influence of childhood maltreatment in these samples (Sinclair et al. 2012;Sinclair et al. 2012B).

We hypothesized that the level of CM in patients with BD was associated with differences in expression of genes involved in the HPA axis functioning. For this purpose, we measured the mRNA levels of nine HPA axis genes in lymphoblastoid cell lines from patients with BD according to their self-reported exposure to CM and using disjoint graph analyses.

Material and Methods

Study sample:

The sample consisted of 33 Caucasian euthymic patients with a diagnosis of BD type 1 according to DSM-IV criteria, from a multicentric cohort (Clinical Trials Number NCT02627404) that investigated the genetic and environmental factors of vulnerability in BD.

The ethical committee (Comité de Protection des Personnes -La Pitié-Salpétrière hospital -Paris -France -reference: P111002-IDRCB2008-AO1465-50) approved the study. All participants provided written informed consent prior to inclusion. Details on inclusion and exclusion criteria have been described previously [START_REF] Etain | Preferential Association between Childhood Emotional Abuse and Bipolar Disorder[END_REF].

The exposure to CM was assessed with the Childhood Trauma Questionnaire (CTQ) that characterizes the presence and severity of five subtypes of CM: emotional abuse (EA), emotional neglect (EN), physical abuse (PA), physical neglect (PN) and sexual abuse (SA) [START_REF] Bernstein | Initial Reliability and Validity of a New Retrospective Measure of Child Abuse and Neglect[END_REF]. The CTQ also generates a total score that is recommended to be used as a continuous variable corresponding to the global exposure to CM (range 25-125).

2.2 Biological sample preparation: Lymphoblastoid cell lines (LCLs) were cultured in RPMI-1640 medium containing 2 mM of L-glutamine, 10 % fetal bovine serum and 1 % penicillin/streptomycin (Life Technologies, France) in a 5 % CO2 humidified incubator at 37 °C. LCLs were seeded at 2×10 5 cells/ml. After 4 days, cells were harvested for RNA isolation. Total RNA was extracted from 5×10 6 cells pellets using the miRNeasy Mini Kit according to the manufacturer's protocol (QIAGEN, France) and quantified with a NanoDrop One spectrophotometer (ThermoFisher Scientific, France). Total RNAs were stored at -80 °C until processing.

Quantitative RT-PCR:

1 μg of total RNA was reverse transcribed, in a final volume of 25 μl, using the iScript™ Reverse Transcription Supermix following the manufacturer's protocol (Bio-Rad laboratories, France). After reverse transcription, cDNAs were stored at -20 °C. Custom designed 384 wells Prime PCR plates were used. Plates were pre-plated with 16 genes of the HPA pathways and 5 reference genes (Bio-Rad laboratories, France; supplementary Table A).

SsoAvanced Universal SYBR Green Supermix (Bio-Rad laboratories) was used for amplification following the manufacturer's instructions. The amplification was performed on a 7900HT instrument (Thermofischer). The specificity of PCR products was verified using a melting curve analysis step. Assays were carried out in duplicate. GAPDH, SDHA and HPRT1 were selected as reference genes using built-in GeNorm analysis of the CFX Maestro software (Biorad). Baseline correction and threshold setting were performed using automatic calculation. Expression levels of 7 genes (CRH, CRHBP, CRHR1, CRHR2, UCN2, UCN3, and NR3C2) were too low to be analyzed. We studied the expression of the 9 following genes: DGKH, FKBP5, NR3C1, SGK1, SGK2, SGK3, SKA2, STAT5A and UCN.

Statistical analyses:

All analyses were performed using R version 3.5.1. RT-qPCR data were analyzed using the method of disjoint graphs gene networks, described in [START_REF] Curis | Determination of Sets of Covariating Gene Expression Using Graph Analysis on Pairwise Expression Ratios[END_REF], as implemented in the SARP.compo package version 0.9.0 for R. Briefly, all pairwise gene expression ratios are compared between two conditions, using a Student test on log-transformed ratios. A graph is built using these tests results: each node is a gene, and two nodes are linked if the corresponding ratio does not change significantly between the two conditions. Difference in gene expression is then defined as disjoint sets of genes. To ensure a Type I error of incorrectly observing a disconnected graph less than 5 %, the individual cut-off for a given ratio was set to 0.23 based on 5 000 simulations (14 nodes, for two groups of 16 and 17 subjects respectively; simulation results: 95 % confidence interval [0.2395; 0.2610]). Here the conditions being studied in association with gene expression were: 1) the global level of CM measured by the quantitative total score of the CTQ and 2) the presence of CM subtypes based on the binary classification of severity, using the cut-offs for "absent or low CM" versus "moderate or severe CM" as described in [START_REF] Bernstein | Initial Reliability and Validity of a New Retrospective Measure of Child Abuse and Neglect[END_REF].

Results

All patients (n=33) presented with a BD type 1 diagnosis, with a mean age of onset of BD around 28 years, and a mean duration of illness around 19 years. They were mainly women (57.6%). The mean CTQ total score was 40,33 +/-12,04 (range: 25-83). When considering the presence of CM subtypes (defined by a moderate or severe levels), we observed the following frequencies: EN= 24 %, EA= 15 %, PN= 27 %, PA= 9 % and SA= 9 %. Further details are given in supplementary Table B.

We first explored the association between the global level of childhood maltreatment and levels of transcripts. We found no correlations between CTQ total score and amount of HPA axis transcripts (see correlogram in Supplementary Figure A). This was then confirmed by the network analysis using SARP.compo (with the CTQ total score as a continuous variable) that did not show any association between the CTQ total score and any of the expression level of the genes (Figure A.1).

We then explored the five trauma subtypes. Since only 3 patients (9 %) had a "moderate or severe" SA or PA, analyses using these two scores were considered as unreliable. Hence, PA and SA were not further investigated. Using Wilcoxon rank tests, we found no differences of transcripts levels according to the presence/absence of EN or PN (data available on request). For EA, the univariate analyses using Wilcoxon rank tests (see Table A) identified a decreased expression of two transcripts, DGKH (p = 0.009) and NR3C1 (p = 0.04), in presence of a moderate or severe score of EA.

Finally, for EA (used as dichotomous variable), we confirmed the results of the univariable analysis by a disjoint graphs network analysis that showed two groups of genes being differently co-expressed in presence of EA (Figure A.2). The smallest group consisted of NR3C1 and DGKH genes that, in presence of EA, were not co-expressed anymore (i.e. No further covariate (listed in Supplementary Table C) was included in the graph analysis since we identified no association between EA and these potential confounders.

Discussion

This study investigated the association between CM, and more specifically emotional abuse, and the levels of expression of nine genes of the HPA axis in lymphoblastoid cell lines of euthymic patients with BD type 1. The gene expression covariation network analysis identified, in presence of EA, a modified co-expression of DGKH and NR3C1, as compared to the other genes of the HPA axis. According to the univariate analyses, this modification corresponds to a decreased co-expression of these two transcripts in presence of emotional abuse.

Several previous studies have addressed the issue of interactions between early life adversities and comprehensive sets of HPA axis genes in BD (or related phenotypes), mainly using genetic variants (e.g. [START_REF] Segura | Association of Childhood Trauma and Genetic Variability of CRH-BP and FKBP5 Genes with Suicidal Behavior in Bipolar Patients[END_REF][START_REF] Spijker | Glucocorticoid and Mineralocorticoid Receptor Polymorphisms and Clinical Characteristics in Bipolar Disorder Patients[END_REF]). Hence, this study reinforces the existing literature by further suggesting an association between CM and alterations of HPA axis gene expression. One strength of this study is to use disjoint graph analysis of a comprehensive set of HPA axis genes, and not only focusing on a single gene.

Interestingly, these results are consistent with the literature investigating methylation of HPA axis genes in association with CM. Indeed, several independent studies have reported differential methylation of NR3C1 (mostly in the sense of hypermethylation) in presence of CM leading to transcriptional silencing [START_REF] Watkeys | Glucocorticoid Receptor Gene (NR3C1) DNA Methylation in Association with Trauma, Psychopathology, Transcript Expression, or Genotypic Variation: A Systematic Review[END_REF]. Moreover, NR3C1 has been described as one of the 9 genes whose expression were similarly modulated in blood and brain of "PTSD-like" rats [START_REF] Daskalakis | Expression Profiling Associates Blood and Brain Glucocorticoid Receptor Signaling with Trauma-Related Individual Differences in Both Sexes[END_REF]. According to this latter study, NR3C1 could be one of the best candidates for the study of the effects of CM on gene expression level in blood since it could be representative of the alterations in the brain.

Another strength of this study is to provide a disentanglement of CM, this being feasible thanks to the use of the CTQ. Indeed, a finding of our study is that the total score of CM did not seem to influence HPA genes expression, whereas EA did. This observation deserves several comments. First, a global measure of CM may not be specific or precise enough, or too noisy, to identify the biological correlates. Second, emotional abuse, even if considered as a possible "low-grade" CM (as compared to PA and SA) may be enough to alter biological systems. This result is parallel to those concluding that the effect of EA in BD was particularly robust (Palmier-Clauss 2016). Nevertheless, we cannot exclude also possible contributions of SA and PA but these were not investigated here due to the small number of patients having been exposed. Therefore, further studies are required to extend this disentanglement.

This study has several limitations. First, we included a small number of participants, leading to potential false negative results. The small sample size also implies that we have not been able to use other subtypes of CM in the analyses since the number of individuals with moderate to severe sexual or physical abuse was too small. Second, the assessment of CM has been made using a self-report questionnaire. Although validated in clinical and nonclinical samples, the CTQ may lead to biases in under or over-reporting CM. Nevertheless, it has been reported that the risk of minimization is higher in controls as compared to individuals with psychiatric disorders (with BD, recurrent depression or schizophrenia) [START_REF] Church | Childhood Trauma and Minimization/Denial in People with and without a Severe Mental Disorder[END_REF]. In a sample of individuals with first-episode psychosis and controls, it has been suggested no strong evidence that the validity of the two used measures (CTQ and a comprehensive interview about childhood trauma) differed between cases and controls [START_REF] Gayer-Anderson | A Comparison between Self-Report and Interviewer-Rated Retrospective Reports of Childhood Abuse among Individuals with First-Episode Psychosis and Population-Based Controls[END_REF]. Furthermore, the CTQ does not provide the chronology and repetitive nature of the different CM subtypes an individual has been exposed to. Third, RT-qPCR was performed on lymphoblastoid cell lines, which might not correspond to the mechanisms at stake in the brain. Nevertheless, given the difficulties to access to brain samples of patients, lymphoblastoid cell lines have been proposed as a good model for the identification of biomarkers of brain diseases [START_REF] Wheeler | Lymphoblastoid Cell Lines in Pharmacogenomic Discovery and Clinical Translation[END_REF]. Finally, our results were based on association tests that cannot lead to infer causality.

  excluded from the network) with the other HPA axis genes. Dendograms are shown on supplementary Figure B.

Acknowledgements

This work was supported by INSERM (Institut National de la Santé et de la Recherche Médicale -Research Protocol C0829), Assistance Publique des Hôpitaux de Paris (Research Protocol GAN12). This research was also supported by the "Investissements d'Avenir" program managed by the ANR (Agence Nationale de la Recherche) under reference ANR-11-IDEX-0004 and Fondation FondaMental (RTRS Santé Mentale). We thank INSERM and Labex BioPsy (ANR-10-IAIHU-06) for the PhD funding (Poste Accueil) to DGL. CMC is supported by the Centre National pour la Recherche.

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In conclusion, the joint graph analysis of a set of nine genes involved in the HPA axis functioning suggested a decreased co-expression of DGKH and NR3C1 in association with emotional abuse in euthymic patients with BD. Further studies would be required to elucidate the causal relationships between the exposure to CM and HPA axis dysregulation in BD.