Validating Baboon Ex Vivo and In Vivo Radiation-Related Gene Expression with Corresponding Human Data

Abstract

The research for high-throughput diagnostic tests for victims of radio/nuclear incidents remains ongoing. In this context, we have previously identified candidate genes that predict risk of late-occurring hematologic acute radiation syndrome (HARS) in a baboon model. The goal of the current study was to validate these genes after radiation exposure in humans. We also examined ex vivo relative to in vivo measurements in both species and describe dose-response relationships. Eighteen baboons were irradiated in vivo to simulate different patterns of partial- or total-body irradiation (TBI), corresponding to an equivalent dose of 2.5 or 5 Sv. Human in vivo blood samples were obtained from patients exposed to different dose ranges: diagnostic computerized tomography (CT; 0.004-0.018 Sv); radiotherapy for prostate cancer (0.25-0.3 Sv); and TBI of leukemia patients (2 × 1.5 or 2 × 2 Sv, five patients each). Peripheral whole blood of another five baboons and human samples from five healthy donors were cultivated ex vivo and irradiated with 0-4 Sv. RNA was isolated pairwise before and 24 h after irradiation and converted into cDNA. Gene expression of six promising candidate genes found previously by us in a baboon model ( WNT3, POU2AF1, CCR7, ARG2, CD177, WLS), as well as three genes commonly used in ex vivo whole blood experiments ( FDXR, PCNA, DDB2) was measured using qRT-PCR. We confirmed the six baboon candidate genes in leukemia patients. However, expression for the candidate gene FDXR showed an inverse relationship, as it was downregulated in baboons and upregulated in human samples. Comparisons among the in vivo and ex vivo experiments revealed the same pattern in both species and indicated peripheral blood cells to represent the radiation-responsive targets causing WNT3 and POU2AF1 gene expression changes. CCR7, ARG2, CD177 and WLS appeared to be altered due to radiation-responsive targets other than the whole blood cells. Linear dose-response relationships of FDXR, WNT3 and POU2AF1 using human ex vivo samples corresponded with human in vivo samples, suggesting that ex vivo models for in vivo dose estimates can be used over a wide dose range (0.001-5 Sv for POU2AF1). In summary, we validated six baboon candidate genes in humans, but the FDXR measurements underscored the importance of independent assessments even when candidates from animal models have striking gene sequence homology to humans. Since whole blood cells represented the same radiation-responsive targets for FDXR, WNT3 and POU2AF1 gene expression changes, ex vivo cell culture models can be utilized for in vivo dose estimates over a dose range covering up to 3.5 log scales. These findings might be a step forward in the development of a gene expression-based high-throughput diagnostic test for populations involved in large-scale radio/nuclear incidents.

Persistent mRNA and miRNA expression changes in irradiated baboons

“…Since we used TaqMan chemistry with human primer and probe sequences (high sensitivity and specificity), it is more likely that we lost some of the detectable human RNA species due to a mismatch with the baboon genome, rather than producing false positives. Also, in validation studies using human samples from irradiated patients, we were able to reproduce candidate genes from our previous baboon studies implying that differences in genomic sequences represent a minor problem 29 .…”

Overcoming challenges in human saliva gene expression measurements

“…In the present work, four different TaqMan Gene Expression Assays (CDKN1A, Hs00355782_m1; FDXR, Hs01031617_m1; DDB2, Hs00772068_m1; and GAPDH, Hs00902257_m1) were utilized and pooled to enable the multiplex amplification of specific cDNA targets. Candidate genes were selected based on two criteria: (1) Genes that were known to be expressed in sufficient quantities for gene expression analysis in whole blood and/or (2) genes that were described to be radiation sensitive in previous studies 23,30 . To ensure linearity of the pre-amplification step, the thermal cycling was set to 10 and 14 cycles for each sample in order to show linearity and, thus, an unbiased pre-amplification.…”

“…Our group has extensive experience in the isolation of RNA from whole blood, its cellular subgroups and biopsies of different tissue sources [20][21][22][23][24] . We recently became interested in isolating RNA from saliva.…”

Individual Radiosensitivity in Oncological Patients: Linking Adverse Normal Tissue Reactions and Genetic Features

“…In humans, FDXR expression is upregulated in a dose-dependent manner after irradiation, both ex vivo and in vivo , indicating that FDXR is a good biomarker for radiation exposure and for estimating in vivo dose (46–48). Moreover, FDXR belongs to a genetic signature for the early prediction of hematological acute radiation syndrome (47, 49). Unlike those authors who have found no association between FDXR expression and the hematological acute radiation syndrome in subjects undergoing RT, our work associates FDXR expression level with dermatitis radiation at the completion of RT treatment (G2 vs. G3 grades, adjusted p = 0.096) (Table 3).…”