Impact of Simulated Rotating Shift Work on Mammary Tumor Development in the p53R270H©/+WAPCre Mouse Model for Breast Cancer.

Epidemiological studies associate night shift work with increased breast cancer risk. However, the underlying mechanisms are not clearly understood. To better understand these mechanisms, animal models that mimic the human situation of different aspects of shift work are needed. In this study, we used "timed sleep restriction" (TSR) cages to simulate clockwise and counterclockwise rotating shift work schedules and investigated predicted sleep patterns and mammary tumor development in breast tumor-prone female p53R270H©/+WAPCre mice. We show that TSR cages are effective in disturbing normal activity and estimated sleep patterns. Although circadian rhythms were not shifted, we observed effects of the rotating schedules on sleep timing and sleep duration. Sleep loss during a simulated shift was partly compensated after the shift and also partly during the free days. No effects were observed on body weight gain and latency time of breast cancer development. In summary, our study shows that the TSR cages can be used to model shift work in mice and affect patterns of activity and sleep. The effect of disturbing sleep patterns on carcinogenesis needs to be further investigated.

Corrigendum: The use of progeroid DNA repair-deficient mice for assessing anti-aging compounds, illustrating the benefits of nicotinamide riboside.

[This corrects the article DOI: 10.3389/fragi.2022.1005322.].

The use of progeroid DNA repair-deficient mice for assessing anti-aging compounds, illustrating the benefits of nicotinamide riboside.

Despite efficient repair, DNA damage inevitably accumulates with time affecting proper cell function and viability, thereby driving systemic aging. Interventions that either prevent DNA damage or enhance DNA repair are thus likely to extend health- and lifespan across species. However, effective genome-protecting compounds are largely lacking. Here, we use Ercc1 Δ/- and Xpg -/- DNA repair-deficient mutants as two bona fide accelerated aging mouse models to test propitious anti-aging pharmaceutical interventions. Ercc1 Δ/- and Xpg -/- mice show shortened lifespan with accelerated aging across numerous organs and tissues. Previously, we demonstrated that a well-established anti-aging intervention, dietary restriction, reduced DNA damage, and dramatically improved healthspan, strongly extended lifespan, and delayed all aging pathology investigated. Here, we further utilize the short lifespan and early onset of signs of neurological degeneration in Ercc1 Δ/- and Xpg -/- mice to test compounds that influence nutrient sensing (metformin, acarbose, resveratrol), inflammation (aspirin, ibuprofen), mitochondrial processes (idebenone, sodium nitrate, dichloroacetate), glucose homeostasis (trehalose, GlcNAc) and nicotinamide adenine dinucleotide (NAD+) metabolism. While some of the compounds have shown anti-aging features in WT animals, most of them failed to significantly alter lifespan or features of neurodegeneration of our mice. The two NAD+ precursors; nicotinamide riboside (NR) and nicotinic acid (NA), did however induce benefits, consistent with the role of NAD+ in facilitating DNA damage repair. Together, our results illustrate the applicability of short-lived repair mutants for systematic screening of anti-aging interventions capable of reducing DNA damage accumulation.

Unlike dietary restriction, rapamycin fails to extend lifespan and reduce transcription stress in progeroid DNA repair-deficient mice.

Dietary restriction (DR) and rapamycin extend healthspan and life span across multiple species. We have recently shown that DR in progeroid DNA repair-deficient mice dramatically extended healthspan and trippled life span. Here, we show that rapamycin, while significantly lowering mTOR signaling, failed to improve life span nor healthspan of DNA repair-deficient Ercc1∆/- mice, contrary to DR tested in parallel. Rapamycin interventions focusing on dosage, gender, and timing all were unable to alter life span. Even genetically modifying mTOR signaling failed to increase life span of DNA repair-deficient mice. The absence of effects by rapamycin on P53 in brain and transcription stress in liver is in sharp contrast with results obtained by DR, and appoints reducing DNA damage and transcription stress as an important mode of action of DR, lacking by rapamycin. Together, this indicates that mTOR inhibition does not mediate the beneficial effects of DR in progeroid mice, revealing that DR and rapamycin strongly differ in their modes of action.

Time-restricted feeding improves adaptation to chronically alternating light-dark cycles.

Disturbance of the circadian clock has been associated with increased risk of cardio-metabolic disorders. Previous studies showed that optimal timing of food intake can improve metabolic health. We hypothesized that time-restricted feeding could be a strategy to minimize long term adverse metabolic health effects of shift work and jetlag. In this study, we exposed female FVB mice to weekly alternating light-dark cycles (i.e. 12 h shifts) combined with ad libitum feeding, dark phase feeding or feeding at a fixed clock time, in the original dark phase. In contrast to our expectations, long-term disturbance of the circadian clock had only modest effects on metabolic parameters. Mice fed at a fixed time showed a delayed adaptation compared to ad libitum fed animals, in terms of the similarity in 24 h rhythm of core body temperature, in weeks when food was only available in the light phase. This was accompanied by increased plasma triglyceride levels and decreased energy expenditure, indicating a less favorable metabolic state. On the other hand, dark phase feeding accelerated adaptation of core body temperature and activity rhythms, however, did not improve the metabolic state of animals compared to ad libitum feeding. Taken together, restricting food intake to the active dark phase enhanced adaptation to shifts in the light-dark schedule, without significantly affecting metabolic parameters.

The transcriptomic response to irinotecan in colon carcinoma bearing mice preconditioned by fasting.

BACKGROUND: Irinotecan use is limited due to severe toxicity. Preconditioning by fasting (PBF) protects against side effects of irinotecan while preserving its antitumor activity. The mechanisms underlying the effects of PBF still need to be elucidated. Here, we investigated the transcriptional responses of PBF on irinotecan in both tumor and healthy liver tissue. EXPERIMENTAL APPROACH: Male BALB/c mice were subcutaneously injected with C26 colon carcinoma cells. Twelve days after tumor inoculation, two groups were fasted for three days and two groups were allowed food ad libitum (AL). Subsequently, both groups received one dose of irinotecan. Twelve hours after administration mice were sacrificed and blood, tumor and liver tissue were harvested. Blood samples were analyzed to determine liver, kidney and bone marrow function, tissues were used for transcriptome analyses. KEY RESULTS: The AL irinotecan group showed worsened organ function and decreased leukocyte numbers. These effects were abated in PBF animals. PBF led to an altered transcriptional response in the liver of irinotecan-treated mice, including decreased cellular injury and increased stress resistance. Hepatic metabolism of irinotecan was also significantly changed due to PBF. The transcriptional response of tumor tissue observed after PBF was hardly affected compared to AL fed animals. CONCLUSIONS: Transcriptional changes after PBF to irinotecan treatment showed an improved protective stress response in healthy liver but not in tumor tissue, including changes in irinotecan metabolism. These data help to unravel the mechanisms underlying the effects of fasting on irinotecan and help to improve outcome of chemotherapeutic treatment in cancer patients.

Antioxidants linked with physical, cognitive and psychological frailty: Analysis of candidate biomarkers and markers derived from the MARK-AGE study.

Frailty among elderly people leads to an increased risk for negative health outcomes. To prevent frailty, we need a better understanding of the underlying mechanisms and early detection of individuals at risk. Both may be served by identifying candidate (bio)markers, i.e. biomarkers and markers, for the physical, cognitive, and psychological frailty domains. We used univariate (Rank-ANOVA) and multivariate (elastic net) approaches on the RASIG study population (age range: 35-74 years, n = 2220) of the MARK-AGE study to study up to 331 (bio)markers between individuals with and without frailty for each domain. Biomarkers and markers identified by both approaches were studied further regarding their association with frailty using logistic regression. Univariately, we found lower levels of antioxidants, including ß-cryptoxanthin and zeaxanthin, in those who were physically, cognitively or psychologically frail. Additionally, self-reported health was worse in these three frail groups. Multivariately, we observed lower levels of ß-cryptoxanthin and zeaxanthin in the cognitively frail. Levels of these carotenoids were inversely associated with the risk of being cognitively frail after adjusting for confounders. Antioxidants and self-reported health are potential (bio)markers to detect persons at risk of becoming frail. The biomarkers identified may indicate the involvement of inflammation in frailty, especially for physical and cognitive frailty.

Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1Δ/- mice.

DNA damage is an important contributor to endothelial dysfunction and age-related vascular disease. Recently, we demonstrated in a DNA repair-deficient, prematurely aging mouse model (Ercc1Δ/- mice) that dietary restriction (DR) strongly increases life- and health span, including ameliorating endothelial dysfunction, by preserving genomic integrity. In this mouse mutant displaying prominent accelerated, age-dependent endothelial dysfunction we investigated the signaling pathways involved in improved endothelium-mediated vasodilation by DR, and explore the potential role of the renin-angiotensin system (RAS). Ercc1Δ/- mice showed increased blood pressure and decreased aortic relaxations to acetylcholine (ACh) in organ bath experiments. Nitric oxide (NO) signaling and phospho-Ser1177-eNOS were compromised in Ercc1Δ/- DR improved relaxations by increasing prostaglandin-mediated responses. Increase of cyclo-oxygenase 2 and decrease of phosphodiesterase 4B were identified as potential mechanisms. DR also prevented loss of NO signaling in vascular smooth muscle cells and normalized angiotensin II (Ang II) vasoconstrictions, which were increased in Ercc1Δ/- mice. Ercc1Δ/- mutants showed a loss of Ang II type 2 receptor-mediated counter-regulation of Ang II type 1 receptor-induced vasoconstrictions. Chronic losartan treatment effectively decreased blood pressure, but did not improve endothelium-dependent relaxations. This result might relate to the aging-associated loss of treatment efficacy of RAS blockade with respect to endothelial function improvement. In summary, DR effectively prevents endothelium-dependent vasodilator dysfunction by augmenting prostaglandin-mediated responses, whereas chronic Ang II type 1 receptor blockade is ineffective.

Short-Term Preoperative Calorie and Protein Restriction Is Feasible in Healthy Kidney Donors and Morbidly Obese Patients Scheduled for Surgery.

INTRODUCTION: Surgery-induced oxidative stress increases the risk of perioperative complications and delay in postoperative recovery. In mice, short-term preoperative dietary and protein restriction protect against oxidative stress. We investigated the feasibility of a calorie- and protein-restricted diet in two patient populations. METHODS: In this pilot study, 30 live kidney donors and 38 morbidly obese patients awaiting surgery were randomized into three groups: a restricted diet group, who received a synthetic liquid diet with 30% fewer calories and 80% less protein for five consecutive days; a group who received a synthetic diet containing the daily energy requirements (DER); and a control group. Feasibility was assessed using self-reported discomfort, body weight changes, and metabolic parameters in blood samples. RESULTS: Twenty patients (71%) complied with the restricted and 13 (65%) with the DER-diet. In total, 68% of the patients reported minor discomfort that resolved after normal eating resumed. The mean weight loss on the restricted diet was significantly greater (2.4 kg) than in the control group (0 kg, p = 0.002), but not in the DER-diet (1.5 kg). The restricted diet significantly reduced levels of serum urea and plasma prealbumin (PAB) and retinol binding protein (RBP). CONCLUSIONS: A short-term preoperative calorie- and protein-restricted diet is feasible in kidney donors and morbidly obese patients. Compliance is high and can be objectively measured via changes in urea, PAB, and RBP levels. These results demonstrate that this diet can be used to study the effects of dietary restriction on surgery-induced oxidative stress in a clinical setting.

Immunization with Small Amyloid-ß-derived Cyclopeptide Conjugates Diminishes Amyloid-ß-Induced Neurodegeneration in Mice.

BACKGROUND: Soluble oligomeric (misfolded) species of amyloid-ß (Aß) are the main mediators of toxicity in Alzheimer's disease (AD). These oligomers subsequently form aggregates of insoluble fibrils that precipitate as extracellular and perivascular plaques in the brain. Active immunization against Aß is a promising disease modifying strategy. However, eliciting an immune response against Aß in general may interfere with its biological function and was shown to cause unwanted side-effects. Therefore, we have developed a novel experimental vaccine based on conformational neo-epitopes that are exposed in the misfolded oligomeric Aß, inducing a specific antibody response. OBJECTIVE: Here we investigate the protective effects of the experimental vaccine against oligomeric Aß1-42-induced neuronal fiber loss in vivo. METHODS: C57BL/6 mice were immunized or mock-immunized. Antibody responses were measured by enzyme-linked immunosorbent assay. Next, mice received a stereotactic injection of oligomeric Aß1-42 into the nucleus basalis of Meynert (NBM) on one side of the brain (lesion side), and scrambled Aß1-42 peptide in the contralateral NBM (control side). The densities of choline acetyltransferase-stained cholinergic fibers origination from the NBM were measured in the parietal neocortex postmortem. The percentage of fiber loss in the lesion side was determined relative to the control side of the brain. RESULTS: Immunized responders (79%) showed 23% less cholinergic fiber loss (p = 0.01) relative to mock-immunized mice. Moreover, fiber loss in immunized responders correlated negatively with the measured antibody responses (R2 = 0.29, p = 0.02). CONCLUSION: These results may provide a lead towards a (prophylactic) vaccine to prevent or at least attenuate (early onset) AD symptoms.

TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts.

3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.

The Relationship Between Shift Work and Metabolic Risk Factors: A Systematic Review of Longitudinal Studies.

CONTEXT: Although the metabolic health effects of shift work have been extensively studied, a systematic synthesis of the available research is lacking. This review aimed to systematically summarize the available evidence of longitudinal studies linking shift work with metabolic risk factors. EVIDENCE ACQUISITION: A systematic literature search was performed in 2015. Studies were included if (1) they had a longitudinal design; (2) shift work was studied as the exposure; and (3) the outcome involved a metabolic risk factor, including anthropometric, blood glucose, blood lipid, or blood pressure measures. EVIDENCE SYNTHESIS: Eligible studies were assessed for their methodologic quality in 2015. A best-evidence synthesis was used to draw conclusions per outcome. Thirty-nine articles describing 22 studies were included. Strong evidence was found for a relation between shift work and increased body weight/BMI, risk for overweight, and impaired glucose tolerance. For the remaining outcomes, there was insufficient evidence. CONCLUSIONS: Shift work seems to be associated with body weight gain, risk for overweight, and impaired glucose tolerance. Overall, lack of high-methodologic quality studies and inconsistency in findings led to insufficient evidence in assessing the relation between shift work and other metabolic risk factors. To strengthen the evidence, more high-quality longitudinal studies that provide more information on the shift work schedule (e.g., frequency of night shifts, duration in years) are needed. Further, research to the (mediating) role of lifestyle behaviors in the health effects of shift work is recommended, as this may offer potential for preventive strategies.

Whole chromosome aneuploidy in the brain of Bub1bH/H and Ercc1-/Δ7 mice.

High levels of aneuploidy have been observed in disease-free tissues, including post-mitotic tissues such as the brain. Using a quantitative interphase-fluorescence in situ hybridization approach, we previously reported a chromosome-specific, age-related increase in aneuploidy in the mouse cerebral cortex. Increased aneuploidy has been associated with defects in DNA repair and the spindle assembly checkpoint, which in turn can lead to premature aging. Here, we quantified the frequency of aneuploidy of three autosomes in the cerebral cortex and cerebellum of adult and developing brain of Bub1b(H/H) mice, which have a faulty mitotic checkpoint, and Ercc1(-/Δ7) mice, defective in nucleotide excision repair and inter-strand cross-link repair. Surprisingly, the level of aneuploidy in the brain of these murine models of accelerated aging remains as low as in the young adult brains from control animals, i.e. <1% in the cerebral cortex and ∼0.1% in the cerebellum. Therefore, based on aneuploidy, these adult mice with reduced life span and accelerated progeroid features are indistinguishable from age-matched, normal controls. Yet, during embryonic development, we found that Bub1b(H/H), but not Ercc1(-/Δ7) mice, have a significantly higher frequency of aneuploid nuclei relative to wild-type controls in the cerebral cortex, reaching a frequency as high as 40.3% for each chromosome tested. Aneuploid cells in these mutant mice are likely eliminated early in development through apoptosis and/or immune-mediated clearance mechanisms, which would explain the low levels of aneuploidy during adulthood in the cerebral cortex of Bub1b(H/H) mice. These results shed light on the mechanisms of removal of aneuploidy cells in vivo.

Diurnal Variation of Hormonal and Lipid Biomarkers in a Molecular Epidemiology-Like Setting.

INTRODUCTION: Many molecular epidemiology studies focusing on high prevalent diseases, such as metabolic disorders and cancer, investigate metabolic and hormonal markers. In general, sampling for these markers can occur at any time-point during the day or after an overnight fast. However, environmental factors, such as light exposure and food intake might affect the levels of these markers, since they provide input for the internal time-keeping system. When diurnal variation is larger than the inter-individual variation, time of day should be taken into account. Importantly, heterogeneity in diurnal variation and disturbance of circadian rhythms among a study population might increasingly occur as a result of our increasing 24/7 economy and related variation in exposure to environmental factors (such as light and food). AIM: The aim of the present study was to determine whether a set of often used biomarkers shows diurnal variation in a setting resembling large molecular epidemiology studies, i.e., non-fasted and limited control possibilities for other environmental influences. RESULTS: We show that markers for which diurnal variation is not an issue are adrenocorticotropic hormone, follicle stimulating hormone, estradiol and high-density lipoprotein. For all other tested markers diurnal variation was observed in at least one gender (cholesterol, cortisol, dehydroepiandrosterone sulfate, free fatty acids, low-density lipoprotein, luteinizing hormone, prolactin, progesterone, testosterone, triglycerides, total triiodothyronine and thyroid-stimulating hormone) or could not reliably be detected (human growth hormone). DISCUSSION: Thus, studies investigating these markers should take diurnal variation into account, for which we provide some options. Furthermore, our study indicates the need for investigating diurnal variation (in literature or experimentally) before setting up studies measuring markers in routine and controlled settings, especially since time-of-day likely matters for many more markers than the ones investigated in the present study.

Chronically Alternating Light Cycles Increase Breast Cancer Risk in Mice.

Although epidemiological studies in shift workers and flight attendants have associated chronic circadian rhythm disturbance (CRD) with increased breast cancer risk, causal evidence for this association is lacking. Several scenarios have been proposed to contribute to the shift work-cancer connection: (1) internal desynchronization, (2) light at night (resulting in melatonin suppression), (3) sleep disruption, (4) lifestyle disturbances, and (5) decreased vitamin D levels due to lack of sunlight. The confounders inherent in human field studies are less problematic in animal studies, which are therefore a good approach to assess the causal relation between circadian disturbance and cancer. However, the experimental conditions of many of these animal studies were far from the reality of human shift workers. For example, some involved xenografts (addressing tumor growth rather than cancer initiation and/or progression), chemically induced tumor models, or continuous bright light exposure, which can lead to suppression of circadian rhythmicity. Here, we have exposed breast cancer-prone p53(R270H/+)WAPCre conditional mutant mice (in a FVB genetic background) to chronic CRD by subjecting them to a weekly alternating light-dark (LD) cycle throughout their life. Animals exposed to the weekly LD inversions showed a decrease in tumor suppression. In addition, these animals showed an increase in body weight. Importantly, this study provides the first experimental proof that CRD increases breast cancer development. Finally, our data suggest internal desynchronization and sleep disturbance as mechanisms linking shift work with cancer development and obesity.

Biomarkers for circadian rhythm disruption independent of time of day.

Frequent shift work causes disruption of the circadian rhythm and might on the long-term result in increased health risk. Current biomarkers evaluating the presence of circadian rhythm disturbance (CRD), including melatonin, cortisol and body temperature, require 24-hr ("around the clock") measurements, which is tedious. Therefore, these markers are not eligible to be used in large-scale (human) studies. The aim of the present study was to identify universal biomarkers for CRD independent of time of day using a transcriptomics approach. Female FVB mice were exposed to six shifts in a clockwise (CW) and counterclockwise (CCW) CRD protocol and sacrificed at baseline and after 1 shift, 6 shifts, 5 days recovery and 14 days recovery, respectively. At six time-points during the day, livers were collected for mRNA microarray analysis. Using a classification approach, we identified a set of biomarkers able to classify samples into either CRD or non-disrupted based on the hepatic gene expression. Furthermore, we identified differentially expressed genes 14 days after the last shift compared to baseline for both CRD protocols. Non-circadian genes differentially expressed upon both CW and CCW protocol were considered useful, universal markers for CRD. One candidate marker i.e. CD36 was evaluated in serum samples of the CRD animals versus controls. These biomarkers might be useful to measure CRD and can be used later on for monitoring the effectiveness of intervention strategies aiming to prevent or minimize chronic adverse health effects.

Controlled induction of DNA double-strand breaks in the mouse liver induces features of tissue ageing.

DNA damage has been implicated in ageing, but direct evidence for a causal relationship is lacking, owing to the difficulty of inducing defined DNA lesions in cells and tissues without simultaneously damaging other biomolecules and cellular structures. Here we directly test whether highly toxic DNA double-strand breaks (DSBs) alone can drive an ageing phenotype using an adenovirus-based system based on tetracycline-controlled expression of the SacI restriction enzyme. We deliver the adenovirus to mice and compare molecular and cellular end points in the liver with normally aged animals. Treated, 3-month-old mice display many, but not all signs of normal liver ageing as early as 1 month after treatment, including ageing pathologies, markers of senescence, fused mitochondria and alterations in gene expression profiles. These results, showing that DSBs alone can cause distinct ageing phenotypes in mouse liver, provide new insights in the role of DNA damage as a driver of tissue ageing.

Mammary gland tumor promotion by chronic administration of IGF1 and the insulin analogue AspB10 in the p53R270H/⁺WAPCre mouse model.

INTRODUCTION: Insulin analogues are structurally modified molecules with altered pharmaco-kinetic and -dynamic properties compared to regular human insulin used by diabetic patients. While these compounds are tested for undesired mitogenic effects, an epidemiological discussion is ongoing regarding an association between insulin analogue therapy and increased cancer incidence, including breast cancer. Standard in vivo rodent carcinogenesis assays do not pick up this possible increased carcinogenic potential. METHODS: Here we studied the role of insulin analogues in breast cancer development. For this we used the human relevant mammary gland specific p53R270H/⁺WAPCre mouse model. Animals received life long repeated treatment with four different insulin (-like) molecules: normal insulin, insulin glargine, insulin X10 (AspB10) or insulin-like growth factor 1 (IGF1). RESULTS: Insulin-like molecules with strong mitogenic signaling, insulin X10 and IGF1, significantly decreased the time for tumor development. Yet, insulin glargine and normal insulin, did not significantly decrease the latency time for (mammary gland) tumor development. The majority of tumors had an epithelial to mesenchymal transition phenotype (EMT), irrespective of treatment condition. Enhanced extracellular signaling related kinase (Erk) or serine/threonine kinase (Akt) mitogenic signaling was in particular present in tumors from the insulin X10 and IGF1 treatment groups. CONCLUSIONS: These data indicate that insulin-like molecules with enhanced mitogenic signaling increase the risk of breast cancer development. Moreover, the use of a tissue specific cancer model, like the p53R270H/⁺WAPCre mouse model, is relevant to assess the intrinsic pro-carcinogenic potential of mitogenic and non-mitogenic biologicals such as insulin analogues.

TP53 mutations induced by BPDE in Xpa-WT and Xpa-Null human TP53 knock-in (Hupki) mouse embryo fibroblasts.

Somatic mutations in the tumour suppressor gene TP53 occur in more than 50% of human tumours; in some instances exposure to environmental carcinogens can be linked to characteristic mutational signatures. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalization assay (HIMA) is a useful model for studying the impact of environmental carcinogens on TP53 mutagenesis. In an effort to increase the frequency of TP53-mutated clones achievable in the HIMA, we generated nucleotide excision repair (NER)-deficient HUFs by crossing the Hupki mouse with an Xpa-knockout (Xpa-Null) mouse. We hypothesized that carcinogen-induced DNA adducts would persist in the TP53 sequence of Xpa-Null HUFs leading to an increased propensity for mismatched base pairing and mutation during replication of adducted DNA. We found that Xpa-Null Hupki mice, and HUFs derived from them, were more sensitive to the environmental carcinogen benzo[a]pyrene (BaP) than their wild-type (Xpa-WT) counterparts. Following treatment with the reactive metabolite of BaP, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), Xpa-WT and Xpa-Null HUF cultures were subjected to the HIMA. A significant increase in TP53 mutations on the transcribed strand was detected in Xpa-Null HUFs compared to Xpa-WT HUFs, but the TP53-mutant frequency overall was not significantly different between the two genotypes. BPDE induced mutations primarily at G:C base pairs, with approximately half occurring at CpG sites, and the predominant mutation type was G:C>T:A in both Xpa-WT and Xpa-Null cells. Further, several of the TP53 mutation hotspots identified in smokers' lung cancer were mutated by BPDE in HUFs (codons 157, 158, 245, 248, 249, 273). Therefore, the pattern and spectrum of BPDE-induced TP53 mutations in the HIMA are consistent with TP53 mutations detected in lung tumours of smokers. While Xpa-Null HUFs exhibited increased sensitivity to BPDE-induced damage on the transcribed strand, NER-deficiency did not enhance TP53 mutagenesis resulting from damage on the non-transcribed strand in this model.