High information content assays for genetic toxicology testing: A report of the International Workshops on Genotoxicity Testing (IWGT).

We live in an era of 'big data', where the volume, velocity, and variety of the data being generated is increasingly influencing the way toxicological sciences are practiced. With this in mind, a workgroup was formed for the 2017 International Workshops on Genotoxicity Testing (IWGT) to consider the use of high information content data in genetic toxicology assessments. Presentations were given on adductomics, global transcriptional profiling, error-reduced single-molecule sequencing, and cellular phenotype-based assays, which were identified as methodologies that are relevant to present-day genetic toxicology assessments. Presenters and workgroup members discussed the state of the science for these methodologies, their potential use in genetic toxicology, current limitations, and the future work necessary to advance their utility and application. The session culminated with audience-assisted SWOT (strength, weakness, opportunities, and threats) analyses. The summary report described herein is structured similarly. A major conclusion of the workgroup is that while conventional regulatory genetic toxicology testing has served the public well over the last several decades, it does not provide the throughput that has become necessary in modern times, and it does not generate the mechanistic information that risk assessments ideally take into consideration. The high information content assay platforms that were discussed in this session, as well as others under development, have the potential to address aspect(s) of these issues and to meet new expectations in the field of genetic toxicology.

ADP-ribosylation of guanosine by SCO5461 protein secreted from Streptomyces coelicolor.

The Streptomyces coelicolor A3(2) genome encodes a possible secretion protein, SCO5461, that shares a 30% homology with the activity domains of two toxic ADP-ribosyltransferases, pierisins and mosquitocidal toxin. We found ADP-ribosylating activity for the SCO5461 protein product through its co-incubation with guanosine and NAD(+), which resulted in the formation of N(2)-(ADP-ribos-1-yl)-guanosine ((ar2)Guo), with a K(m) value of 110 µM. SCO5461 was further found to ADP-ribosylate deoxyguanosine, GMP, dGMP, GTP, dGTP, and cyclic GMP with k(cat) values of 150-370 s(-1). Oligo(dG), oligo(G), and yeast tRNA were also ADP-ribosylated by this protein, although with much lower k(cat) values of 0.2 s(-1) or less. SCO5461 showed maximum ADP-ribosylation activity towards guanosine at 30 °C, and maintained 20% of these maximum activity levels even at 0 °C. This is the first report of the ADP-ribosylation of guanosine and guanine mononucleotides among the family members of various ADP-ribosylating enzymes. We additionally observed secretion of the putative gene product, SCO5461, in liquid cultures of S. coelicolor. We thus designated the SCO5461 protein product as S. coelicolor ADP-ribosylating protein, ScARP. Our current results could offer new insights into not only the ADP-ribosylation of small molecules but also signal transduction events via enzymatic nucleoside modification by toxin-related enzymes.

Molecular cloning of apoptosis-inducing Pierisin-like proteins, from two species of white butterfly, Pieris melete and Aporia crataegi.

Pierisin-1, present in cabbage butterfly, Pieris rapae, induces apoptosis against various kinds of cancer cell lines. Another cabbage butterfly, Pieris brassicae, also has an apoptosis-inducing protein, Pierisin-2. These proteins exhibit DNA ADP-ribosylating activity. Pierisin-like proteins are found to be distributed in subtribes Pierina, Aporiina and Appiadina. In this study, we performed the cDNA cloning of Pierisin-like proteins designated Pierisin-3 from gray-veined white, Pieris melete, and Pierisin-4 from black-veined white, Aporia crataegi. The nucleotide sequences of Pierisin-3 and -4 encode an 850 and an 858 amino acid protein, respectively. The partial peptide sequences of Pierisin-3 and -4 purified from pupae were identical to the deduced amino acid sequence of ORF. The deduced amino acid sequence revealed that Pierisin-3 is 93% similar to Pierisin-1 and Pierisin-4 is 64%. Pierisin-3 and -4 synthesized in vitro with the rabbit reticulocyte lysate exhibited apoptosis-inducing activity against human cervical carcinoma HeLa and human gastric carcinoma TMK-1 cells. Site-directed mutagenesis at a glutamic acid residue comprising the NAD-binding site resulted in a significant decrease in cytotoxicity of both proteins. Moreover, the proteins incubated with calf thymus DNA and beta-NAD resulted in the formation of N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine, as in the case of Pierisin-1 and -2. These findings could provide useful information for understanding the importance of apoptosis-inducing ability and molecular evolution of Pierisin-like proteins in family Pieridae.