Development of mammary cancer in γ-irradiated F1 hybrids of susceptible Sprague-Dawley and resistant Copenhagen rats, with copy-number losses that pinpoint potential tumor suppressors.

Copenhagen rats are highly resistant to mammary carcinogenesis, even after treatment with chemical carcinogens and hormones; most studies indicate that this is a dominant genetic trait. To test whether this trait is also dominant after radiation exposure, we characterized the susceptibility of irradiated Copenhagen rats to mammary carcinogenesis, as well as its inheritance, and identified tumor-suppressor genes that, when inactivated or mutated, may contribute to carcinogenesis. To this end, mammary cancer-susceptible Sprague-Dawley rats, resistant Copenhagen rats, and their F1 hybrids were irradiated with 4 Gy of γ-rays, and tumor development was monitored. Copy-number variations and allelic imbalances of genomic DNA were studied using microarrays and PCR analysis of polymorphic markers. Gene expression was assessed by quantitative PCR in normal tissues and induced mammary cancers of F1 rats. Irradiated Copenhagen rats exhibited a very low incidence of mammary cancer. Unexpectedly, this resistance trait did not show dominant inheritance in F1 rats; rather, they exhibited intermediate susceptibility levels (i.e., between those of their parent strains). The susceptibility of irradiated F1 rats to the development of benign mammary tumors (i.e., fibroadenoma and adenoma) was also intermediate. Copy-number losses were frequently observed in chromosome regions 1q52-54 (24%), 2q12-15 (33%), and 3q31-42 (24%), as were focal (38%) and whole (29%) losses of chromosome 5. Some of these chromosomal regions exhibited allelic imbalances. Many cancer-related genes within these regions were downregulated in mammary tumors as compared with normal mammary tissue. Some of the chromosomal losses identified have not been reported previously in chemically induced models, implying a novel mechanism inherent to the irradiated model. Based on these findings, Sprague-Dawley × Copenhagen F1 rats offer a useful model for exploring genes responsible for radiation-induced mammary cancer, which apparently are mainly located in specific regions of chromosomes 1, 2, 3 and 5.

Exome of Radiation-induced Rat Mammary Carcinoma Shows Copy-number Losses and Mutations in Human-relevant Cancer Genes.

BACKGROUND/AIM: Our understanding of cancer risk from neutron exposure is limited. We aimed to reveal the characteristics of mammary carcinomas induced by neutrons. MATERIALS AND METHODS: Mammary carcinomas obtained from female Sprague-Dawley rats irradiated at 7 weeks of age with 0.97 Gy neutrons or 4 Gy γ-rays and from non-irradiated rats were classified into luminal and non-luminal subtypes by immunohistochemistry. Their mutational landscapes were determined by whole-exome sequencing. RESULTS: Neutrons significantly raised the incidence of luminal mammary carcinomas over the non-luminal subtype. Somatic mutations were identified in cancer genes involved in several signalling pathways, including Keap1/Nrf2, Pi3k/Akt and Wnt/ß-catenin. Focal copy-number losses involving cancer genes were observed mainly in carcinomas from the irradiated rats. CONCLUSION: Neutrons increase the incidence of luminal mammary carcinomas, probably through gene mutations similar to those found in human breast cancers, and focal copy-number losses including cancer genes that are characteristics of radiation-induced mammary carcinomas.

Neutron-induced Rat Mammary Carcinomas Are Mainly of Luminal Subtype and Have Multiple Copy Number Aberrations.

BACKGROUND/AIM: Neutrons are used as a type of high linear energy transfer (LET) radiation and they have stronger carcinogenic effects compared to low LET radiation. We sought to clarify the features of mammary carcinomas for which the incidence increases when these were exposed to neutron radiation. MATERIALS AND METHODS: We compared mammary carcinomas from female Sprague-Dawley rats irradiated at 7 weeks of age with 0.485 Gy neutron beams or 0.5-Gy γ rays, with carcinomas of non-irradiated rats. Tumors were classified into luminal and non-luminal subtypes based on immunohistochemistry, while their copy number aberrations were determined using microarrays. RESULTS: Neutrons and γ rays significantly increased the incidence of luminal carcinomas. The carcinomas in the three groups contained multiple aberrations affecting 46 genes for which mutations have been reported in human breast cancer. CONCLUSION: Neutrons and γ rays increase the incidence of luminal mammary carcinoma in rats, probably via genetic aberrations similar to those found in human breast cancer patients.

Risk of second cancer after ion beam radiotherapy: insights from animal carcinogenesis studies.

Purpose: To review recent studies to better understand the risk of second cancer after ion beam radiotherapy and to clarify the importance of animal radiobiology therein. Results: Risk of developing second cancer after radiotherapy is a concern, particularly for survivors of childhood tumors. Ion beam radiotherapy is expected to reduce the risk of second cancer by reducing exposure of normal tissues to radiation. Large uncertainty lies, however, in the choice of relative biological effectiveness (RBE) of high linear energy transfer (LET) radiation (e.g. carbon ions and neutrons) in cancer induction, especially for children. Studies have attempted to predict the risk of second cancer after ion beam radiotherapy based on an assessment of radiation dose, the risk of low LET radiation, and assumptions about RBE. Animal experiments have yielded RBE values for selected tissues, radiation types, and age at the time of irradiation; the results indicate potentially variable RBE which depends on tissues, ages, and dose levels. Animal studies have also attempted to identify genetic alterations in tumors induced by high LET radiation. Conclusions: Estimating the RBE value for cancer induction is important for understanding the risk of second cancer after ion beam radiotherapy. More comprehensive animal radiobiology studies are needed.

Differential effect of parity on rat mammary carcinogenesis after pre- or post-pubertal exposure to radiation.

Radiation exposure during the peri-pubertal period is a proven risk factor for breast cancer, whereas parity is an established protective factor. The present study investigated whether parity imposes differential protective effects against radiation-induced rat mammary carcinoma depending on the age at exposure. Pre- and post-pubertal female rats, irradiated or left unirradiated, were mated and allowed to nurse until weaning or left unmated. Appearance of mammary tumors was monitored, and serum concentrations of estradiol and progesterone were measured following weaning. Carcinomas were evaluated by immunohistochemistry for estrogen receptor, progesterone receptor, and the cell proliferation marker Ki-67. Parity reduced the risk of carcinoma in unirradiated and pre-pubertally irradiated rats but not post-pubertally irradiated rats. Although radiation exposure increased serum progesterone level, parity after pre-pubertal exposure significantly decreased the elevated progesterone to a normal level, reflecting a protective effect. Moreover, parity significantly decreased the proportion of hormone receptor-positive carcinomas after pre-pubertal exposure. Parity was also related to the observed positive association between progesterone receptor and Ki-67 indices in cancer tissue, implying progesterone receptor-dependent cell proliferation. Thus, parity protects against radiation-induced rat mammary carcinogenesis depending on the age at exposure; the mechanisms may involve changes in hormone levels and cancer tissue.

Age Modifies the Effect of 2-MeV Fast Neutrons on Rat Mammary Carcinogenesis.

The relative biological effectiveness (RBE) of neutrons depends on their physical nature (e.g., energy) and the biological context (e.g., end points, materials). From the perspective of radiological protection, age is an important biological context that influences radiation-related cancer risk, but very few studies have addressed its potential impact on neutron effects. We therefore investigated the influence of age on the effect of accelerator-generated fast neutrons (mean energy, ∼2 MeV) in an animal model of breast carcinogenesis. Female Sprague-Dawley rats at 1, 3 and 7 weeks of age were irradiated with fast neutrons at absorbed doses of 0.0485-0.97 Gy. All animals were kept under specific pathogen-free conditions and screened weekly for mammary tumors by palpation until they were 90 weeks old. Tumors were diagnosed based on histology. Mathematical modeling was used to analyze mammary cancer incidence, collectively using data from this study and a previously reported experiment on 137Cs gamma rays. The results indicate that neutron irradiation elevated the risk of palpable mammary carcinoma with a linear dose response, the slope of which depended on age at time of irradiation. The RBE of neutron radiation was 7.5 ± 3.4, 9.3 ± 3.5 and 26.1 ± 8.9 (mean ± SE) for animals exposed at 1, 3 and 7 weeks of age, respectively. Our results indicate that age of the animal is an important factor influencing the effect of fast neutrons on breast cancer risk.

Analysis of genes involved in the PI3K/Akt pathway in radiation- and MNU-induced rat mammary carcinomas.

The PI3K/AKT pathway is one of the most important signaling networks in human breast cancer, and since it was potentially implicated in our preliminary investigations of radiation-induced rat mammary carcinomas, our aim here was to verify its role. We included mammary carcinomas induced by the chemical carcinogen 1-methyl-1-nitrosourea to determine whether any changes were radiation-specific. Most carcinomas from both groups showed activation of the PI3K/AKT pathway, but phosphorylation of AKT1 was often heterogeneous and only present in a minority of carcinoma cells. The negative pathway regulator Inpp4b was significantly downregulated in both groups, compared with in normal mammary tissue, and radiation-induced carcinomas also showed a significant decrease in Pten expression, while the chemically induced carcinomas showed a decrease in Pik3r1 and Pdk1. Significant upregulation of the positive regulators Erbb2 and Pik3ca was observed only in chemically induced carcinomas. However, no genes showed clear correlations with AKT phosphorylation levels, except in individual carcinomas. Only rare carcinomas showed mutations in PI3K/AKT pathway genes, yet these carcinomas did not exhibit stronger AKT phosphorylation. Thus, while AKT phosphorylation is a common feature of rat mammary carcinomas induced by radiation or a canonical chemical carcinogen, the mutation of key genes in the pathways or permanent changes to gene expression of particular signaling proteins do not explain the pathway activation in the advanced cancers. Although AKT signaling likely facilitates cancer development and growth in rat mammary carcinomas, it is unlikely that permanent disruption of the PI3K/AKT pathway genes is a major causal event in radiation carcinogenesis.

A novel in vitro pharmacokinetic/pharmacodynamic model based on two-compartment open model used to simulate serum drug concentration-time profiles.

An in vitro pharmacokinetic/pharmacodynamic perfusion model that simulates a two-compartment open model of serum drug concentration-time profiles following intravenous bolus injection and infusion was developed and mathematically described. In the present apparatus model, flow was kept in a one-way mode to avoid liquid traffic, and the washout effect seen in dilution models was overcome by embedding the tested bacteria in low melting point agarose gel. The validity of the equations and the reproducibility of the apparatus model were ascertained by simulating the concentration-time profiles of cefazolin and fosfomycin by substitution of their pharmacokinetic parameters obtained from humans for the equations. An empirical regimen 1X(q24h) of 1 g with cefazolin administered by intravenous infusion effectively killed a Staphylococcus aureus strain. The same regimen with fosfomycin produced a marked kill-curve with a fosfomycin-susceptible enterohaemorrhagic Escherichia coli O157:H7, whereas considerable regrowth was observed with a resistant strain. These results indicated that the present model was able to provide a convenient and reliable method for evaluating the efficacy of antimicrobial agents administered by intravenous infusion.