7,12-Dimethylbenz(a)anthracene-induced genotoxicity on bone marrow cells from mice phenotypically selected for low acute inflammatory response

Exposure to polycyclic aromatic hydrocarbon (PAH) environmental contaminants has been associated with the development of mutations and cancer. 7,12-Dimethylbenz(a)anthracene ( DMBA), a genotoxic agent, reacts with DNA directly, inducing p53-dependent cytotoxicity resulting in cell death by apoptosis or giving rise to cancer. DMBA metabolism largely depends on activation of the aryl hydrocarbon receptor (AhR). Mice phenotypically selected for high (AIRmax) or low (AIRmin) acute inflammatory response present a complete segregation of Ahr alleles endowed with low (Ahr(d)) or high (Ahr(b1)) affinity to PAHs, respectively. To evaluate the role of AhR genetic polymorphism on the bone marrow susceptibility to DMBA, AIRmax and AIRmin mice were treated with a single intraperitoneal injection of DMBA (50 mg/kg b.w.) in olive oil. Bone marrow cells (BMCs) were phenotyped by both flow cytometry and cytoslide preparations. Despite a significant decrease in total cell count in BM from AIRmin mice, there was an increase of blast cells and immature neutrophils at 1 and 50 days after DMBA treatment, probably due to a cell-cycle blockade at the G1/S transition leading to immature stage cell production. A panel of proteins related to cell cycle regulation was evaluated in immature BM cells (Lin(-)) by Western Blot, and DNA damage and repair were measured using an alkaline version of the Comet assay. In Lin(-) cells isolated from AIRmin mice, high levels were found in both p53 and p21 protein contents in contrast with the low levels of CDK4 and Ciclin D1. Evaluation of DNA repair in DMBA-treated BMCs, indicated long-lasting genotoxicity and cytotoxicity in BMC from AIRmin mice and a blockade of cell cycle progression. On the other hand, AIRmax mice have a high capacity of DNA damage repair and protection. These mechanisms can be associated with the differential susceptibility to the toxic and carcinogenic effects of DMBA observed in these mice. (C) 2015 Elsevier B.V. All rights reserved.

Distinct gene expression profiles provoked by polyacrylamide beads (Biogel) during chronic and acute inflammation in mice selected for maximal and minimal inflammatory responses

AIRmax and AIRmin mice differ in their local acute inflammatory reactions to polyacrylamide beads (Biogel). These lines were developed to identify genes that affect the intensity of the acute inflammatory response (AIR) and to investigate the cellular and molecular mechanisms of acute inflammation. Although these lines are well established, differences in their responses to chronic inflammatory Biogel exposure have not yet been described. We investigated whether the selective process that modified the acute inflammatory responses in these animals also affected the development of their chronic inflammatory responses. Inflammatory exudate cell infiltration was more intense in AIRmax than AIRmin mice at both 48 h and 30 days. Genes involved in signal transduction and immune/inflammatory responses were differentially expressed in the treated skin of AIRmax and AIRmin mice, and divergent expression of some acute inflammatory response genes was detected up to 30 days post-Biogel. However, distinct expression of several pro and anti-inflammatory response genes in both periods was observed. These results indicate that the selective process for acute inflammation affected the development of chronic inflammatory responses to Biogel, suggesting common genetic control

Genetic linkage analysis identifies Pas1 as the common locus modulating lung tumorigenesis and acute inflammatory response in mice.

Selective breeding for the acute inflammatory response (AIR) generated two mouse lines characterized by maximum (AIRmax) and minimum (AIRmin) responses, explained by the additive effect of alleles differentially fixed in quantitative trait loci (QTLs). These mice also differ in their susceptibility to lung tumorigenesis, raising the possibility that the same loci are involved in the control of both phenotypes. To map the QTLs responsible for the different phenotypes, we carried out a genome-wide linkage analysis using single-nucleotide polymorphism arrays in a pedigree consisting of 802 mice, including 693 (AIRmax × AIRmin)F2 intercross mice treated with urethane and phenotyped for AIR and lung tumor multiplicity. We mapped five loci on chromosomes 4, 6, 7, 11 and 13 linked to AIR (logarithm of odds (LOD)=3.56, 3.52, 15.74, 7.74 and 3.34, respectively) and two loci linked to lung tumor multiplicity, on chromosomes 6 and 18 (LOD=12.18 and 4.69, respectively). The known pulmonary adenoma susceptibility 1 (Pas1) locus on chromosome 6 was the only locus linked to both phenotypes, suggesting that alleles of this locus were differentially fixed during breeding and selection of AIR mice. These results represent a step toward understanding the link between inflammation and cancer.

Association study by genetic clustering detects multiple inflammatory response loci in non-inbred mice

We tested the possibility to map loci affecting the acute inflammatory response (AIR) in an (AIRmax AIRmin) F2 intercrossmouse population derived from non-inbred parents, by association analysis in the absence of pedigree information. Using 1064 autosomal single nucleotide polymorphisms (SNPs), we clustered the intercross population into 12 groups of genetically related individuals. Association analysis adjusted for genetic clusters allowed to identify two loci, inflammatory response modulator 1 (Irm1) on chromosome 7 previously detected by genetic linkage analysis in the F2 mice, and a new locus onchromosome 5 (Irm2), linked to the number of infiltrating cells in subcutaneous inflammatory exudates (Irm1: P»6.3 10 7; Irm2: P»8.2 10 5) and interleukin 1 beta (IL-1b) production (Irm1: P»1.9 10 16; Irm2: P»1.1 10 6). Use of a polygenic model based on additive effects of the rare alleles of 15 or 18 SNPs associated at suggestive genome-wide statistical threshold(Po3.4 10 3) with the number of infiltrating cells or IL-1b production, respectively, allowed prediction of the inflammatory response of progenitor AIR mice. Our findings suggest the usefulness of association analysis in combination with genetic clustering to map loci affecting complex phenotypes in non-inbred animal species.

Association study by genetic clustering detects multiple inflammatory response loci in non-inbred mice.

We tested the possibility to map loci affecting the acute inflammatory response (AIR) in an (AIRmax × AIRmin) F2 intercross mouse population derived from non-inbred parents, by association analysis in the absence of pedigree information. Using 1064 autosomal single nucleotide polymorphisms (SNPs), we clustered the intercross population into 12 groups of genetically related individuals. Association analysis adjusted for genetic clusters allowed to identify two loci, inflammatory response modulator 1 (Irm1) on chromosome 7 previously detected by genetic linkage analysis in the F2 mice, and a new locus on chromosome 5 (Irm2), linked to the number of infiltrating cells in subcutaneous inflammatory exudates (Irm1: P=6.3 × 10(-7); Irm2: P=8.2 × 10(-5)) and interleukin 1 beta (IL-1ß) production (Irm1: P=1.9 × 10(-16); Irm2: P=1.1 × 10(-6)). Use of a polygenic model based on additive effects of the rare alleles of 15 or 18 SNPs associated at suggestive genome-wide statistical threshold (P<3.4 × 10(-3)) with the number of infiltrating cells or IL-1ß production, respectively, allowed prediction of the inflammatory response of progenitor AIR mice. Our findings suggest the usefulness of association analysis in combination with genetic clustering to map loci affecting complex phenotypes in non-inbred animal species.

A new model of outbred genetically selected mice which present a strong acute inflammatory response in the absence of complement component C5.

OBJECTIVE: Mice selected for a strong (AIRmax) or weak (AIRmin) acute inflammatory response present different susceptibilities to bacterial infections, autoimmune diseases and carcinogenesis. Variations in these phenotypes have been also detected in AIRmax and AIRmin mice rendered homozygous for Slc11a1 resistant (R) and susceptible (S) alleles. Our aim was to investigate if the phenotypic differences observed in these mice was related to the complement system. MATERIAL: AIRmax and AIRmin mice and AIRmax and AIRmin groups homozygous for the resistance (R) or susceptibility (S) alleles of the solute carrier family 11a1 member (Slc11a1) gene, formerly designated Nramp-1. METHODS AND RESULTS: While no difference in complement activity was detected in sera from AIRmax and AIRmin strains, all sera from AIRmax Slc11a1 resistant mice (AIRmax(RR)) presented no complement-dependent hemolytic activity. Furthermore, C5 was not found in their sera by immunodiffusion and, polymerase chain reaction and DNA sequencing of its gene demonstrated that AIRmax(RR) mice are homozygous for the C5 deficient (D) mutation previously described in A/J. Therefore, the C5D allele was fixed in homozygosis in AIRmax(RR) line. CONCLUSIONS: The AIRmax(RR) line is a new experimental mouse model in which a strong inflammatory response can be triggered in vivo in the absence of C5.