In the last half century, various methods such as the Ames test and the micronucleus test have been employed as simple, sensitive, and reliable approaches for evaluation of the mutagenicity of chemicals and drugs (OECD, 2016; OECD, 2020). More recently, the technological advancement in science has allowed us, in addition to evaluation of mutagenicity, to address the distinct spectra of mutations produced by a broad range of intrinsic and environmental mutagens or anticancer drugs (David, 2020). The mutational signatures extracted from these mutation spectra have provided valuable information about the role of endogenous and exogenous risk factors in the development of many human disorders and have been crucial in finding potential targets for treatment (Nik-Zainal et al., 2012; Meier and Gartner, 2014; Koh et al., 2021).

Until the present day, a vast amount of diverse studies focused on the mechanisms of mutagenesis and DNA repair from the chemical, biochemical, and biological aspects have heavily relied on experimental strategies using cloned genes (Razzaque et al., 1983; Drinkwater and Klinedinst, 1986; Gossen et al., 1989; White et al., 2019). One such widely used system is the SV40-based shuttle vector mutagenesis assay, which utilizes vector plasmids containing the supF gene of Escherichia coli and an indicator bacteria strain (Parris and Seidman, 1992; Parris et al., 1994; Sarkar et al., 1984; Yoon et al., 2019; Suzuki et al., 2022). The supF gene codes for an amber suppressor transfer RNA (tRNA) that translates the amber stop codon (UAG) to tyrosine (Camakaris and Pittard, 1973). Depending upon the position and type of mutations in the supF gene, the supF tRNA cannot translate selectable marker genes with an amber mutation in its indicator E. coli strains, such as KS40/pOF105 (Normanly et al., 1986; Obata et al., 1998). Because of its properties, the supF gene is particularly useful for acquiring mutation spectra and identifying mutation signatures, and provides reliable data on mutations generated by specific DNA lesions on the leading or the lagging template DNA strand via translesion synthesis (TLS) (Drinkwater and Klinedinst, 1986). However, one needs to be aware of the limitation of the phenotypic mutation spectrum of the supF gene; in our experiments for example, from the entire base sequence of the supF gene only about 30% can be detected for dual-antibiotic selection of indicator E. coli, that is, whether these sites can be identified or not is largely dependent on the experimental conditions, including the scale of the experiment, reporter genes for the selection, the mutagens used, the frequency of mutations, and the efficiency of the detection (Obata et al., 1998; Kraemer and Seidman, 1989; Fukushima et al., 2020). Recently, we reported the development of a new indicator E. coli strain – RF01, that is able to identify mutation spectra more efficiently than other strains used for the supF assay, even though it still does not allow the elucidation of trinucleotide mutational signatures because of the limited resolution of the mutation spectrum analysis (Fukushima et al., 2020).These limitations can be overcome by advanced techniques, namely next-generation sequencing (NGS), which over the last decade is enabling us to obtain various kinds of information from nucleic acid sequence data, including mutations and three-dimensional organization of the genomic DNA at a single cell level (Shendure et al., 2017; Dutta et al., 2022; Arrastia et al., 2022). On the other hand, the NGS technology also has its limitations, especially for identification and quantification of low-frequency mutations, as it can be difficult to distinguish true mutations from the baseline errors of NGS. In recent years, in order to detect low-frequency mutations, numerous library construction methods and sequencing methods have been developed, such as maximum depth sequencing or duplex sequencing (Schmitt et al., 2012; Jee et al., 2016; Sloan et al., 2018). For every scientific objective, trade-offs between accuracy, cost, and speed of the multiple available approaches must be carefully considered. Importantly, there is a scarcity of methods whose technical simplicity and material availability enables any researchers, not only those specialized in NGS, to accumulate, reproduce, and compare data across research fields. Certainly, the availability of an easy, low-cost assay for accurate analysis of mutational signatures would benefit many laboratories who are deterred by the complexity of the existing methods.

In this study, we attempt to develop a simple mutagenesis assay using a random 12-nucleotide (12-nt) DNA-barcoded supF shuttle vector library with non-SOS-induced E. coli which can amplify the DNA with ‘a high-fidelity replication mechanism’ as a template for the NGS sample preparation (Napolitano et al., 1997). This promotes cost-benefit, high-throughput, and high accuracy with almost no sequencing errors. In the first part, we present data from the multiplexed NGS assay utilizing the barcoded supF shuttle vector library and the BGI-platform that uses the rolling circle replication method, and we compare the results for different conditions of sample preparation. In the later part of this study, we present analysis of UV radiation-induced mutations in direct E. coli experiments or after transfection into mammalian cells, as an example of the performance of our newly developed assay. UV radiation is a known environmental carcinogen and causes a characteristic mutational pattern via the formation of pyrimidine-pyrimidine dimers (Varghese and Wang, 1967; Mao et al., 2016; Lindberg et al., 2019). The C to T transition at CCN and TCN trinucleotide sites (the mutated base is underlined) is a mutational signature highly specific to UV-mediated mutagenesis (mutational COSMIC signatures SBS7a-d) (Alexandrov et al., 2020). The cytosine in UV-induced pyrimidine-pyrimidine dimers undergoes deamination to uracil, followed by insertion of an A nucleotide opposite the lesion by error-prone TLS polymerases (Pfeifer et al., 2005; Ikehata and Ono, 2011; Roberts and Gordenin, 2014). The substantial amount of sequence data on UV-induced mutations in mammalian cells obtained in this study allowed us to uncover some insightful information about UV-induced mutagenesis, underscoring the importance of a systematic process for sufficient data collection and analysis. We believe that our new approach using the barcoded supF shuttle vector library and NGS technology presents a benefit to future research on mutagenesis, DNA repair, and cancer.

Ever since the 1980s, the supF forward mutagenesis assay which utilizes a supF gene encoding shuttle vector and an indicator E. coli has been widely employed in studies on mutagenicity in E. coli or mammalian cells (Yoon et al., 2019; Camakaris and Pittard, 1973; Kraemer and Seidman, 1989; Seidman et al., 1985; Yoon et al., 2021b). This conventional supF assay is very useful for analysis of both mutation frequencies and mutation spectra. The mutation spectra of single or tandem base substitutions for inactive supF genes identified by using the blue-white colony color assays were comprehensively summarized in an earlier review article, and it was noted that the mutations were detected at 86 sites within a 158 bp region covering the supF gene (54%) and at 74 sites within the 85 bp mature tRNA region (87%), thus demonstrating the great sensitivity of the supF assay system for analysis of mutation spectra (Kraemer and Seidman, 1989). However, obtaining reliable datasets by the conventional supF assay requires skill and experience, especially for studies where the mutations of interest are induced with low frequency. The method has been advanced by the construction of indicator bacterial strains with different supF reporter genes which allow selection based on survival of bacteria containing mutant supF genes. However, the fact that the supF phenotypic selection process relies on the structure and function of transfer RNAs that may be differently affected by different mutations means that the improvement of the efficiency of the selection process may cause loss of coverage of the mutation spectra, as it is under our experimental conditions, where the coverage is about 30% (Kraemer and Seidman, 1989; Fukushima et al., 2020). Since in recent years NGS has become a routine research tool due to its lowering cost, numerous sophisticated applications of NGS technologies are being developed for research in genomics, epigenomics, and transcriptomics (McCombie et al., 2019; Wang et al., 2016). In this study, we attempted to combine the supF shuttle vector forward mutagenesis assay with sequencing analysis by a short-read NGS platform and consequently developed a novel supF NGS assay with potential application in mutagenesis and DNA repair studies. The combination of the DNA-barcoded supF shuttle vector library and its amplification via in vivo replication in the indicator non-SOS E. coli provides us with proper templates for the PCR-facilitated preparation of multiplexed NGS samples. Thus, during the PCR process the amplification and index tagging of the samples are achieved simultaneously. Designed in such a way, the assay allows us to obtain almost all mutation sequences from a small amount of data from NGS reads, especially in samples from colonies grown on selection plates, where the mutation sequences, although prone to bias, are highly concentrated. This results in the possibility to simultaneously analyze hundreds of samples from different experiments in a single NGS run. Here, the cost of mutation spectrum analysis from selection plates was less than $1.00 per sample, even though both the NGS library preparation and the NGS run were outsourced. Further cost reductions can be expected by obtaining more sequencing data in a single run, and also from the continuous improvement of NGS technologies. From this point of view, we believe that we can secure a sufficient number of experiments to improve the accuracy of the analysis and to confirm the reproducibility of the experiments. Furthermore, the data from colonies grown on titer plates provides us, at least in principle, and with the exception of large deletions and insertions, with non-biased mutation spectra for the whole analyzed region. Also, mutation frequencies calculated from titer plate data were 15–20 times higher than those obtained by the conventional supF assay, demonstrating that our novel assay allows us to analyze more thoroughly the mutation spectrum by using a smaller amount of data and to detect mutations with lower frequencies. The analysis of data from both selection and titer plates is consistent with the desired reliability and low cost of the mutagenesis assay. Last but not least, by using the supF shuttle vector plasmid, mutation spectrum data can be obtained with different experimental designs, such as pre-treatment with metabolic enzymes or using cells with different genetic backgrounds, siRNA transfection, CRISPR-Cas systems, etc. We are currently conducting experiments with an supF library containing site-specific single DNA damages such as 8-oxo-7,8-dihydroguanine (8-oxoG, 8-hydroxyguanine) among others (Suzuki et al., 2018; Suzuki et al., 2021; Suzuki et al., 2022), which will allow us to analyze in detail the repair mechanisms of sequence-specific DNA damage and provide further insights into mutagenesis and DNA repair processes.

The current study posits that when performing the assay, we have to be aware of the following points: (1) The required barcode complexity defined by the number of barcode sequences in the supF shuttle vector library depends on the experimental purpose and the mutation detection frequencies. A barcode complexity of 1.0×10⁴ for each library is used as the standard in this paper. Even though more complexity may be able to provide better results, especially at lower mutation frequencies, one must keep in mind that the number of colonies to be harvested for the NGS sample preparation, as well as the platform or setting for data analysis, need to be adjusted depending on the number of barcodes. (2) The number of NGS reads required for each multiplexed sample and for each N₁₂-BC sequence need to be calculated before the preparation of the samples. Therefore, the number of N₁₂-BC sequences and the number of harvested colonies must be optimized first. In addition, the allele frequency given by VarDict, referred to as VF in this study, for each classified N₁₂-BC sequence is the most important determinant of the quality of the variant sequence data and the basis for analyzing overlapping N₁₂-BC sequences. The cost, time, and data quality must be all well considered and balanced when an experiment is designed. (3) NGS data contain sequencing errors, the rate of which is known to be between 10^–3 and 10^–4, with the majority of the errors being A:T  to G:C and G:C to A:T transitions that had occurred during the PCR in the process of NGS library preparation (Brodin et al., 2013; Costello et al., 2013). In the assay presented here no sonication step is required, thus avoiding DNA oxidation. As already mentioned, the clonal amplification of the supF shuttle vector via in vivo replication in the non-SOS E. coli provides a proper PCR template for preparation of NGS samples. In addition, the sequence alignment to the reference sequence for all identical N₁₂-BC sequences and the analysis of data obtained from duplicate PCR reactions can almost completely eliminate the sequencing errors resulting from the process of sample preparation. On the other hand, in the case of the amplicon sequencing employed here, the base-calling errors occurred depending on the ‘per base sequence quality’ of the specific sequence. In this study, in addition to the supF gene with its total length of 335 bp being sequenced, 200 bp paired-end sequencing was performed with about 30 bp overlapping sequences, and the resulting sequence was merged together. The ‘per base sequence quality’ in the region between read positions 160 and 200 bp is relatively low compared to other positions. Data shown in this study, as well as other experiments conducted in our laboratory, reveals that two positions in particular – 55 and 69 in the supF gene manifest a very high frequency of variant calls – 20–50% of samples, but with low VF (less than 0.3). These two positions are located at 165–167 (depending on the N₀, N₁, N₂ primers) and 181–183 bp from the end of the forward reads, and at 179–181 and 167–169 bp from the end of the reverse reads. The addition of different numbers of random nucleotides – 0-, 1-, or 2-nt at the end of both primers used for sample preparation can contribute to better ‘per base sequence quality’ in clonal amplicon sequencing. However, positions 55 and 69 are located immediately next to CC:GG sequences (position 55 is located in TTACC:GGTAA and position 69 is located in GTTCC:GGAAC), and the result of variant calling was almost exclusively SNS from 55A:T or 69T:A to C:G. Thus, these variant calls appear to be position-specific and sequence-dependent, and for this assay can be regarded as position-specific base-calling errors. Since they had a VF of less than 0.3 and a threshold VF of 0.4 was applied in this study, these base-calling errors do not need to be given consideration. Nevertheless, it must be emphasized that fine-tuning must be done for each individual experiment. (4) Large alterations in the supF gene, such as a large deletion or insertion, cannot be analyzed by this assay because of impossibility for PCR amplification or read alignments. This may render our observations on mutation frequency misleading. The frequency of such large alterations is better assessed by colony PCR, especially if the number of colonies grown on the titer plates was different from expected. (5) For the supF assay, spontaneous cluster mutations at TC:GA sites were often observed, and it was well illustrated in an earlier study that a nick in the shuttle vector was a trigger for these asymmetric cluster mutations (Seidman et al., 1987). Therefore, we need to be aware of the quality of each library and how it affects the outcome of each analysis, especially for detection of very low levels of mutations. Depending on the purpose of the experiments, in the preparation of covalently closed circular vector libraries, it is essential to eliminate the background level of mutations. In fact, the in vitro construction of the library of double-stranded shuttle vectors from single-stranded circular DNA requires the process of treatment with T5 exonuclease, which drastically decreases background mutations.

In this study, to test the performance of our newly developed supF NGS assay, we conducted experiments using N₁₂-BC libraries irradiated with UV-C at different doses. Although these pilot experiments were designed in a relatively simple way, they clearly exemplify the mutation spectrum associated with UV radiation exposure, and further experiments may provide profound insights into UV mutagenesis. Here, by using this novel assay we were able to confirm in both E. coli and human cells that the SNSs induced by UV were mainly C:G to T:A transitions, which is a known UV signature, and that the UV-induced DNA lesions were basically dipyrimidines – CPDs at T-T sites, and (6-4)PPs at T-T and T-C sites (Varghese and Wang, 1967; Mao et al., 2016; Lindberg et al., 2019; Kamiya et al., 1993; Kamiya et al., 1998). The SNSs are listed in Figure 10B (presented within pentanucleotide sequences of the supF gene) starting from the most frequent. In samples obtained from titer plates, the nucleotide sequence in eight of the top ten mutational hotspots was TTC/GAA, and the sequence of the top four mutational sites was TTCGA/TCGAA. These top four sites represent two palindromic TTCGAA/TTCGAA hexanucleotide sequences located at positions 115–120 and 123–128 in the supF gene. All top 10 mutations were C to T transitions and occurred at TC sites, and are likely the result of TLS opposite the 3'C of CPDs and (6-4)PPs via specialized DNA polymerases, such as Polη, -ι, -ζ, -κ, -θ, -λ, and Rev1 (Achar and Foiani, 2019; Yoon et al., 2021a). Notably, the site selection bias observed for the top 10 mutation hotspots seems to differ between the titer and the selection plates. In selection plates, three of the top ten hotspot mutations were C to T transitions at the 3'C of CC. These hotspot SNSs, particularly positions 130 and 121, appear to be next to or between hotspot SNSs in the titer plates. However, such SNS mutations, that were only observed in selection plates, were identified in both titer and selection plates as other types of mutations, specifically as hotspot DNS mutations, CC to TT, as shown in Figure 10C (located at 130 and 131 TTCGAATCCTT, at 121 TTCGAAGGTTCGAA, at 5 TTTGATA/TATCAAA, or at 84 and 85 AAGGGAG/CTCCCTT). In addition, as shown in Figure 9B and C, the multiple mutations, either C or G mutations at the same base in identical N₁₂-BC sequences, were identified in a dose-dependent manner more frequently from selection plates where TLS polymerases opposite UV lesions on the same DNA strand can cause clustered mutations. These multiple mutations include at least one phenotypic mutation in the supF gene but include also non-phenotypic mutations, which contributes to the specifics of the mutation spectrum in the selection plates. The two-base substitutions within trinucleotides, referred to as Del-in by VarDict, were mainly detected at higher UV doses as GAG:CTC to AAA:TTT at positions 73–75. It is unclear whether these mutations arose through unexpected UV-induced lesions at GCTCG sites, or the deamination of either the 5'C or the 3'C has occurred at these specific sites by different mechanisms. Alternatively, it can be hypothesized that the UV-induced DNA lesions arose on a single-stranded DNA (ssDNA) followed by a blockage of replication, or during DNA repair processes. It is known that ssDNA can be a substrate for a subclass of activation-induced deaminase/apolipoprotein B mRNA-editing catalytic polypeptide-like subfamily cytosine deaminases (APOBECs), which cause cluster mutations at TC:GA sites in the same strand of DNA by converting cytosine to uracil (Pecori et al., 2022). It is realistic to assume that the mutations at TC:GA sites had occurred via several different mechanisms. In fact, spontaneous mutations in non-UV-irradiated samples transfected into U2OS cells can also be detected at the order of 10^–4 per gene. In both UV-irradiated and non-irradiated samples, SNS mutations had basically occurred from C:G at TC:GA sites. Also, the major proportion of multiple mutations were C:G to T:A transitions. However, in non-UV-irradiated samples, the major proportion of single mutations were C:G to G:C transversions, which differed from the irradiated condition, where the major proportion was G:C to A:T transition (Figure 9C). It is possible that the oxidatively damaged base 8-oxoG was endogenously generated in the U2OS cells. As we have previously reported, 8-oxoG becomes a trigger for the DNA repair process and generates ssDNA as a substrate for APOBECs (Suzuki et al., 2022). Here, the TTC/GAA site has emerged from data from titer plates as a mutational hotspot. According to previous reports, the TTC motif, and more specifically the cytosine in the motif, is a site of preference for APOBEC3A, APOBEC3C, APOBEC3F, and APOBEC3G, even though further investigation is needed (Lindberg et al., 2019; Love et al., 2012; Adolph et al., 2018).

What might be more, a particular mutation was detected by VarDict in non-UV-irradiated samples as a Del-in mutation – a change from TTGAT:ATCAA to ATCAA:TTGAT at positions 3–7 within the potential hairpin loop of a quasi-palindromic sequence (Figure 14E), which may be the consequence of template switching events. The mutation was detected with a frequency at the order of 10^–6 per gene (Figure 12E), and was hardly detected in UV-irradiated samples, where it remains hidden by the UV-introduced mutations. Also, the same mutation was frequently detected as single SNS mutations from T:A to A:T at position 3, from G:C to C:G at position 5, and from T:A to A:T at position 7, or as combinations with other SNS mutations either or both from G:C to A:T at position –3 or/and from T:A to C:G at position 12 (Figure 12—figure supplement 1), which may be dependent on the regions used for template switching. Although the mono- and poly-ubiquitination of PCNA, facilitated by ubiquitin ligases Rad6-Rad18, HLTF, or SHPRH, is involved in the mechanism of switching between TLS and template switching in mammalian cells, the details are still ambiguous (Chiu et al., 2006; Lin et al., 2011; Masuda et al., 2012; Wong et al., 2021). In addition, our recent studies on action-at-a-distance mutations, according to which a site-specific targeted single 8-oxoG or other types of DNA damage cause cluster mutations at untargeted positions, demonstrated a drastic strand bias for mutations at cytosines, specifically at 5’-TC-3’ sites (Suzuki et al., 2018; Suzuki et al., 2021; Suzuki et al., 2022). Data on mutational strand asymmetries from cancer genome sequencing studies have emerged, indicating that APOBEC-associated mutagenesis during replication strongly favors the lagging-strand template (Seplyarskiy et al., 2016; Haradhvala et al., 2016). And most recently, it has been proposed by a study using UPD-seq technique that APOBEC3A targets cytosines in small hairpin loops, within replication forks, in tRNA genes and at transcription start sites (Sakhtemani et al., 2022). In this study, strand biases in base substations from either C or G were clearly observed, consistent with what could be expected from the mechanisms by which UV photolesions are processed, such as cytosine deamination and the involvement of TLS. Interestingly, the strand bias switched between single base substitutions and cluster mutations depending on the doses of UV and the number of mutations per N₁₂-BC. The precise reason and mechanisms behind this remain to be elucidated, but the observation brings new insights into the mechanisms of mutagenesis. In addition, the positions of SNSs in the multiple mutations were closer to each other compared to the theoretically expected positions (Figure 13—figure supplements 2 and 3), which may reflect switching events involving TLS polymerases. It should be noted that the presented data for the distance between two SNSs in the multiple mutations was analyzed from the data from selection plates in order to secure a sufficient number of mutations, and therefore, there may be a bias due to hotspots associated with the selection process. However, the results from the limited number of mutations from the titer plates are similar to these from the selection plates. It can be proposed that this assay may also be applied for analysis of TLS polymerases in mammalian cells.

Figure 14

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Mutational signatures identified in cancer cells are emerging as valuable markers for cancer diagnosis and therapeutics. Innumerable physical, chemical, and biological mutagens, including anticancer drugs, induce characteristic mutations in genomic DNA via specific mutagenic processes. The mutation spectra obtained here by using the presented advanced method were in good agreement with accumulated data from previous papers where the conventional method had been used, with the advantage that our method provided less-biased mutation spectra data. As described above, the datasets presented here highlighted novel mutational signatures and also cluster mutations with a strand bias, which could be associated with the processes of replication, transcription, or repair of DNA damage, including a single-strand break (a nick). In this study, eight series of supF shuttle vector plasmids were constructed, as presented in Figure 1—figure supplement 1; however, the analysis was carried out using N₁₂-BC libraries prepared from either pNGS2-K1 (Figures 1—4) or pNGS2-K3 (Figures 5—10). The pNGS2-K1/-A1/-K4/-A4 and pNGS2-K2/-A2/-K3/-A3 vector series contain an M13 intergenic region with opposite orientations relative to the supF gene, which allows us to incorporate specific types of DNA damage at specific sites in the opposite strand of the vector library. Also, the pNGS2-K1/-A1/-K3/-A3 and pNGS2-K2/-A2/-K4/-A4 vector series contain the SV40 replication origin, which enables bidirectional replication and transcription, at opposite sides of the supF gene. Although this is still preliminary data, it is notable that the spontaneously induced mutations for the different vectors in U2OS cells were not significantly different. Therefore, the here presented mutagenesis assay with NGS, by using these series of libraries, can be applied in many different types of experiments to address both quantitative and qualitative features of mutagenesis. It is possible to design series of libraries containing DNA lesions or sequences suitable for the investigation of specific molecular mechanisms, such as TLS, template switching, and asymmetric cluster mutations. We are currently trying to develop a different test system for broader mutation spectrum analysis using the BC library with random oligonucleotide sequences; however, the assay using the supF gene presented in this paper has a significant advantage in both efficiency as a mutagenesis assay and cost-effectiveness. We believe that the method presented here will be used in further experiments by us and by other laboratories to advance the research on environmental mutagenesis, carcinogenesis, DNA repair, and will hopefully contribute to mutagenesis research by promoting accumulation of data for machine learning.

NGS raw data has been uploaded to NCBI under the accession number PRJDB13753 (DRA014420 for Figures 2 and 3, DRA014421 for Figures 4-7, DRA014422 for Figures 8-11, DRA014422 for the samples without transformation into E. coli).

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Author details

1. Hidehiko Kawai

   1. Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
   2. School of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   kawaih@hiroshima-u.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2213-7166

2. Ren Iwata

   School of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Shungo Ebi

   School of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Ryusei Sugihara

   School of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Shogo Masuda

   Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

6. Chiho Fujiwara

   School of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

7. Shingo Kimura

   Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

8. Hiroyuki Kamiya

   1. Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
   2. School of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – review and editing

   For correspondence

   hirokam@hiroshima-u.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6866-5322

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