Search

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

Local blood flow/oxygen partial pressure (Po2) distributions and flow-Po2 relationships are physiologically relevant. They affect the pathophysiology and treatment of conditions like hemorrhagic shock (HS), but direct noninvasive measures of flow, Po2, and their heterogeneity during prolonged HS are infrequently presented. To fill this void, we report the first quantitative evaluation of flow-Po2 relationships and heterogeneities in normovolemia and during several hours of HS using noninvasive, unbiased, automated acquisition. Anesthetized rats were subjected to tracheostomy, arterial/venous catheterizations, cremaster muscle exteriorization, hemorrhage (40% total blood volume), and laparotomy. Control animals equally instrumented were not subjected to hemorrhage/laparotomy. Every 0.5 h for 4.5 h, noninvasive laser speckle contrast imaging and phosphorescence quenching were employed for nearly 7,000 flow/Po2 measurements in muscles from eight animals, using an automated system. Precise alignment of 16 muscle areas allowed overlapping between flow and oxygenation measurements to evaluate spatial heterogeneity, and repeated measurements were used to estimate temporal heterogeneity. Systemic physiological parameters and blood chemistry were simultaneously assessed by blood samplings replaced with crystalloids. Hemodilution was associated with local hypoxia, but increased flow prevented major oxygen delivery decline. Adding laparotomy and prolonged HS resulted in hypoxia, ischemia, decreased tissue oxygen delivery, and logarithmic flow/Po2 relationships in most regions. Flow and Po2 spatial heterogeneities were higher than their respective temporal heterogeneities, although this did not change significantly over the studied period. This quantitative framework establishes a basis for evaluating therapies aimed at restoring muscle homeostasis, positively impacting outcomes of civilian and military trauma/HS victims. NEW & NOTEWORTHY This is the first study on flow-Po2 relationships during normovolemia, hemodilution, and prolonged hemorrhagic shock using noninvasive methods in multiple skeletal muscle areas of monitored animals. Automated flow/Po2 measurements revealed temporal/spatial heterogeneities, hypoxia, ischemia, and decreased tissue oxygen delivery after trauma/severe hemorrhage. Hemodilution was associated with local hypoxia, but hyperemia prevented a major decline in oxygen delivery. This framework provides a quantitative basis for testing therapeutics that positively impacts muscle homeostasis and outcomes of trauma/hemorrhagic shock victims.

Background Many studies evaluating blood flow and oxygen partial pressure (PO2) do not directly measure both parameters, are confined to few locations/microvessels, and depend on investigator's selection of measuring sites. Moreover, clinically/physiologically relevant systemic parameters are not simultaneously recorded. We implemented an automated system for prolonged blood flow/PO2 acquisition in large areas while collecting relevant systemic information. Methods In anesthetized animals, cardiorespiratory parameters were continuously recorded. Other data were collected at baseline and hourly after 4 hours of hemorrhagic shock. A cremaster muscle was spread over a pedestal fixed to a motorized stage. One 2-dimensional tissue scan allowed 16 noninvasive PO2 measurements using oxygen-dependent phosphorescence quenching and fiber optics. Blood flow was estimated using laser speckle contrast imaging in the same areas used for PO2 measurements. At each timepoint, blood was sampled for extensive biochemistry/coagulation profile. Results The system was used successfully by different operators. A set of flow/PO2 measurements was completed in less than 90 seconds. Muscle flow and PO2 correlated with some but not several systemic parameters, emphasizing the importance of performing both local and systemic evaluations. Conclusion System advantages include integration between local and over 40 systemic parameters, unbiased data collection/analysis, improved performance/sampled area, easy expansion, implementation and maintenance, no customized programming, and simplified training. Combining this system with trauma/prolonged HS models will enhance our ability to investigate tissue stability and select better resuscitation strategies to improve outcomes and survival. Level of evidence Diagnostic test, level V.

Fluorescent light (FL) has been utilized for &asymp, 60 years and has become a common artificial light source under which animals, including humans, spend increasing amounts of time. Although the solar spectrum is quite dissimilar in both wavelengths and intensities, the genetic consequences of FL exposure have not been investigated. Herein, we present comparative RNA-Seq results that establish expression patterns within skin, brain, and liver for Danio rerio, Oryzias latipes, and the hairless mouse (Mus musculus) after exposure to FL. These animals represent diurnal and nocturnal lifestyles, and &asymp, 450 million years of evolutionary divergence. In all three organisms, FL induced transcriptional changes of the acute phase response signaling pathway and modulated inflammation and innate immune responses. Our pathway and gene clustering analyses suggest cellular perception of oxidative stress is promoting induction of primary up-stream regulators IL1B and TNF. The skin and brain of the three animals as well as the liver of both fish models all exhibit increased inflammation and immune responses, however, the mouse liver suppressed the same pathways. Overall, the conserved nature of the genetic responses observed after FL exposure, among fishes and a mammal, suggest the presence of light responsive genetic circuitry deeply embedded in the vertebrate genome.

Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O6-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 μmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O6-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma. Cancer Prev Res; 9(3); 245–52. ©2015 AACR.

The prevalence of hepatocellular carcinoma (HCC) was diminished from 60% to 18% at 15 months of age in C3HeB/FeJ male transgenic mice expressing hMGMT in our previous studies. To directly test if the methyltransferase activity is required for diminished tumor prevalence, two separate lines of transgenic mice bearing an enzymatically inactive form of hMGMT were used. In these lines, cysteine 145 was substituted with alanine (C145A). Expression of the hMGMT C145A transgene in liver was demonstrated by Northern blots and Western blots. Immunohistochemistry revealed predominantly nuclear localization of the hMGMT C145A protein. hMGMT C145A transgenic mice were crossed with lacI transgenic mice to assess mutant frequencies in the presence of the mutant protein. Mutant frequencies were similar among livers of lacI × hMGMT C145A bi-transgenic mice and lacI × wild-type (WT) mice. DNA sequence analysis of recovered lacI mutants revealed similar mutation spectra for hMGMT C145A and WT mice. The prevalence of HCC was also similar for the two tested lines of hMGMT C145A mice, 45% and 48% prevalence with median tumor sizes of 11 and 8 mm, and WT mice, 40% prevalence and median tumor size of 10 mm. These results provide evidence that residue C145 in hMGMT is required to reduce the prevalence of HCC in C3HeB/FeJ mice transgenic for hMGMT. © 2011 Wiley Periodicals, Inc.

The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair (BER), which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential BER enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutant frequency increased earlier in spermatogenic cells from Apex1+/− mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1+/− mice. By old age, mutant frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA (mtDNA) damage revealed an increase in the Apex1+/− spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mtDNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mtDNA integrity in spermatogenic cells throughout life. Mol. Reprod. Dev. 78:906–919, 2011. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.

Spontaneous mutant frequency in the male germline increases with age, thereby increasing the risk of siring offspring with genetic disorders. In the present study we investigated the effect of age on ionizing radiation-induced male germline mutagenesis. lacI transgenic mice were treated with ionizing radiation at 4-, 15- and 26-month-old, and mutant frequencies were determined for pachytene spermatocytes and round spermatids at 15 days or 49 days after ionizing radiation treatment. Cells collected 15 days after treatment were derivatives of irradiated differentiating spermatogenic cells while cells collected 49 days later were derivatives of spermatogonial stem cells. The results showed that (1) spontaneous mutant frequency increased in spermatogenic cells recovered from nonirradiated old mice (26-months-old), particularly in the round spermatids; (2) mutant frequencies were significantly increased in round spermatids obtained from middle-aged mice (15-months-old) and old age mice (26-months-old) at 15 and 49 days after irradiation compared to the sham-treated old mice; and (3) pachytene spermatocytes obtained from 15- or 26-month-old mice displayed a significantly increased mutant frequency at 15 days post irradiation. This study indicates that age modulates the mutagenic response to ionizing radiation in the male germline.

The prevalence of hepatocellular carcinoma (HCC) was diminished from 60% to 18% at 15 months of age in C3HeB/FeJ male transgenic mice expressing hMGMT in our previous studies. To directly test if the methyltransferase activity is required for diminished tumor prevalence, two separate lines of transgenic mice bearing an enzymatically inactive form of hMGMT were used. In these lines, cysteine 145 was substituted with alanine (C145A). Expression of the hMGMT C145A transgene in liver was demonstrated by Northern blots and Western blots. Immunohistochemistry revealed predominantly nuclear localization of the hMGMT C145A protein. hMGMT C145A transgenic mice were crossed with lacI transgenic mice to assess mutant frequencies in the presence of the mutant protein. Mutant frequencies were similar among livers of lacI × hMGMT C145A bi-transgenic mice and lacI × wild-type (WT) mice. DNA sequence analysis of recovered lacI mutants revealed similar mutation spectra for hMGMT C145A and WT mice. The prevalence of HCC was also similar for the two tested lines of hMGMT C145A mice, 45% and 48% prevalence with median tumor sizes of 11 and 8 mm, and WT mice, 40% prevalence and median tumor size of 10 mm. These results provide evidence that residue C145 in hMGMT is required to reduce the prevalence of HCC in C3HeB/FeJ mice transgenic for hMGMT.

The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair, which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential base excision repair enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutation frequency increased earlier in spermatogenic cells from Apex1+/− mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1+/− mice. By old age, mutation frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA damage revealed an increase in the Apex1+/− spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mitochondrial DNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mitochondrial DNA integrity in spermatogenic cells throughout life.