Peto's paradox: Nature has used multiple strategies to keep cancer at bay while evolving long lifespans and large body masses. A systematic mini-review.

Comparative oncology is an understudied field of science. We are far from understanding the key mechanisms behind Peto's paradox, i.e., understanding how long-lived and large animals are not subject to a higher cancer burden despite the longer exposure time to mutations and the larger number of cells exposed. In this work, we investigated the scientific evidence on such mechanisms through a systematic mini-review of the literature about the relation of longevity and/or large body mass with physiological, genetic, or environmental traits among mammalian species. More than forty thousand articles were retrieved from three repositories, and 383 of them were screened using an active-learning-based tool. Of those, 36 articles on longevity and 37 on body mass were selected for the review. Such articles were examined focusing on: number and type of species considered, statistical methods used, traits investigated, and observed relationship with longevity and/or body mass. Where applicable, the traits investigated were matched with one or more hallmarks of cancer. We obtained a list of potential candidate traits to explain Peto's paradox related to replicative immortality, cell senescence, genome instability and mutations, proliferative signaling, growth suppression evasion, and cell resistance to death. Our investigation suggests that different strategies have been followed to prevent cancer in large and long-lived species. The large number of papers retrieved emphasizes that more studies can be launched in the future, using more efficient analytical approaches to comprehensively evaluate the convergent biological mechanisms essential for acquiring longevity and large body mass without increasing cancer risk.

The spontaneous immortalization probability of mammalian cell culture strains, as their proliferative capacity, correlates with species body mass, not longevity.

BACKGROUND: The Peto's paradox consists in the observation that individuals from long-lived and large animal species do not experience a higher cancer incidence, despite being exposed for longer time to the possibility of accumulating mutations and having more target cells exposed to the phenomenon. The existence of this paradox has been recently confirmed (Vincze et al., 2022). Concurrently, robust evidence has been published that longevity involves a convergent evolution of cellular mechanisms that prevent the accumulation of mutations (Cagan et al., 2022). It remains unclear which cellular mechanisms are critical to allow the evolution of a large body mass while keeping cancer at bay. METHODS: Adding to existing data linking cellular replicative potential and species body mass (Lorenzini et al., 2005), we have grown a total of 84 skin fibroblast cell strains from 40 donors of 17 mammalian species and analyzed their Hayflick's limit, i.e., their senescent plateau, and eventual spontaneous immortalization escape. The correlation of immortalization and replicative capacity of the species with their longevity, body mass and metabolism has been assessed through phylogenetic multiple linear regression (MLR). RESULTS: The immortalization probability is negatively related to species body mass. The new evaluation and additional data about replicative potential strengthen our previous observation, confirming that stable and extended proliferation is strongly correlated with the evolution of a large body mass rather than lifespan. CONCLUSION: The relation between immortalization and body mass suggests a need to evolve stringent mechanisms that control genetic stability during the evolution of a large body mass.

Weekday and Weekend Differences in Eating Habits, Physical Activity and Screen Time Behavior among a Sample of Primary School Children: The "Seven Days for My Health" Project.

BACKGROUND: Healthy eating and active lifestyle habits are essential for a child's development, wellbeing, and health. School setting and family environment play a crucial role in shaping these habits and this could be reflected in different behavior patterns during weekdays and weekends. METHODS: We investigated primary school children's lifestyle habits through a cross-sectional analysis of 428 Italian primary school children, with a mean age of 8.99 years (±1.43). Data were collected from May to June 2017 using a weekly diary to assess children's lifestyles. RESULTS: Children who eat their morning snack and lunch at school three or more times during the weekdays were 5.47 times more likely (95% CI 3.02, 10.2) to consume adequate snacks and 7.79 times more likely (95% CI 4.43, 14.5) to have adequate meals than those who did not. CONCLUSION: Consumption of vegetables, lunch, and snacks are significantly more adequate during the weekdays as compared to the weekends. Physical activity levels did not differ between weekdays and weekends. Moreover, children spent more time engaged in physical activities than in front of a screen during both the weekdays and the weekends. The present results are good indicators of the importance of the school canteen in defining correct eating habits. Family-based and school-based interventions could represent valuable integrative strategies for promoting a healthy lifestyle in children.

Unburned Tobacco Cigarette Smoke Alters Rat Ultrastructural Lung Airways and DNA.

INTRODUCTION: Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk. METHODS: Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays. RESULTS: Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression. CONCLUSIONS: Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk. IMPLICATIONS: IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model.

A pro longevity role for cellular senescence.

Cellular senescence is a fundamental process that may play positive or detrimental roles for the organism. It is involved in tissue development and in tumor prevention although during aging is becoming a detrimental process contributing to the decline of tissue functions. In previous investigations, we have uncovered a better capacity to detect DNA damage in cells from long-lived mammals. Here, we report that cultured cells derived from long-lived species have a higher propensity to undergo senescence when challenged with DNA damage than cells derived from short-lived species. Using a panel of cells derived from six mammals, which range in lifespan from 3-4 years up to 120 years, we examined cell cycle response, induction of apoptosis and of cellular senescence. All species exhibited a cell cycle arrest while induction of apoptosis was variable. However, a significant positive correlation was found between the relative percent of cells, within a population which entered senescence following damage, and the lifespan of the species. We suggest that cellular senescence may have a positive role during development allowing it to contribute to the evolution of longevity.

Topical rapamycin reduces markers of senescence and aging in human skin: an exploratory, prospective, randomized trial.

Aging is a major risk factor for the majority of human diseases, and the development of interventions to reduce the intrinsic rate of aging is expected to reduce the risk for age-related diseases including cardiovascular disease, cancer, and dementia. In the skin, aging manifests itself in photodamage and dermal atrophy, with underlying tissue reduction and impaired barrier function. To determine whether rapamycin, an FDA-approved drug targeting the mechanistic target of rapamycin (mTOR) complex, can reduce senescence and markers of aging in human skin, an exploratory, placebo-controlled, interventional trial was conducted in a clinical dermatology setting. Participants were greater than 40 years of age with evidence of age-related photoaging and dermal volume loss and no major morbidities. Thirty-six participants were enrolled in the study, and nineteen discontinued or were lost to follow-up. A significant (P = 0.008) reduction in p16INK4A protein levels and an increase in collagen VII protein levels (P = 0.0077) were observed among participants at the end of the study. Clinical improvement in skin appearance was noted in multiple participants, and immunohistochemical analysis revealed improvement in histological appearance of skin tissue. Topical rapamycin reduced the expression of the p16INK4A protein consistent with a reduction in cellular senescence. This change was accompanied by relative improvement in clinical appearance of the skin and histological markers of aging and by an increase in collagen VII, which is critical to the integrity of the basement membrane. These results indicate that rapamycin treatment is a potential anti-aging therapy with efficacy in humans.Trial registration ClinicalTrials.gov Identifier: NCT03103893.

Co-carcinogenic effects of vitamin E in prostate.

A large number of basic researches and observational studies suggested the cancer preventive activity of vitamin E, but large-scale human intervention trials have yielded disappointing results and actually showed a higher incidence of prostate cancer although the mechanisms underlying the increased risk remain largely unknown. Here we show through in vitro and in vivo studies that vitamin E produces a marked inductive effect on carcinogen-bioactivating enzymes and a pro-oxidant status promoting both DNA damage and cell transformation frequency. First, we found that vitamin E in the human prostate epithelial RWPE-1 cell line has the remarkable ability to upregulate the expression of various phase-I activating cytochrome P450 (CYP) enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), giving rise to supraphysiological levels of reactive oxygen species. Furthermore, our rat model confirmed that vitamin E in the prostate has a powerful booster effect on CYP enzymes associated with the generation of oxidative stress, thereby favoring lipid-derived electrophile spread that covalently modifies proteins. We show that vitamin E not only causes DNA damage but also promotes cell transformation frequency induced by the PAH-prototype benzo[a]pyrene. Our findings might explain why dietary supplementation with vitamin E increases the prostate cancer risk among healthy men.

E-cigarettes induce toxicological effects that can raise the cancer risk.

Electronic cigarettes (e-cigs) are devices designed to deliver nicotine in a vaping solution rather than smoke and without tobacco combustion. Perceived as a safer alternative to conventional cigarettes, e-cigs are aggressively marketed as lifestyle-choice consumables, thanks to few restrictions and a lack of regulatory guidelines. E-cigs have also gained popularity among never-smokers and teenagers, becoming an emergent public health issue. Despite the burgeoning worldwide consumption of e-cigs, their safety remains largely unproven and it is unknown whether these devices cause in vivo toxicological effects that could contribute to cancer. Here we demonstrate the co-mutagenic and cancer-initiating effects of e-cig vapour in a rat lung model. We found that e-cigs have a powerful booster effect on phase-I carcinogen-bioactivating enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), and increase oxygen free radical production and DNA oxidation to 8-hydroxy-2'-deoxyguanosine. Furthermore, we found that e-cigs damage DNA not only at chromosomal level in peripheral blood, such as strand breaks in leucocytes and micronuclei formation in reticulocytes, but also at gene level such as point mutations in urine. Our results demonstrate that exposure to e-cigs could endanger human health, particularly among younger more vulnerable consumers.

DNA Damage Detection by 53BP1: Relationship to Species Longevity.

In order to examine potential differences in genomic stability, we have challenged fibroblasts derived from five different mammalian species of variable longevity with the genotoxic agents, etoposide and neocarzinostatin. We report that cells from longer-lived species exhibit more tumor protein p53 binding protein 1 (53BP1) foci for a given degree of DNA damage relative to shorter-lived species. The presence of a greater number of 53BP1 foci was associated with decreased DNA fragmentation and a lower percentage of cells exhibiting micronuclei. These data suggest that cells from longer-lived species have an enhanced DNA damage response. We propose that the number of 53BP1 foci that form in response to damage reflects the intrinsic capacity of cells to detect and respond to DNA harms.

DNA damage markers in dermal fibroblasts in vitro reflect chronological donor age.

The aging process is accompanied by an accumulation of cellular damage, which compromises the viability and function of cells and tissues. We aim to further explore the association between in vitro DNA damage markers and the chronological age of the donor, as well as long-lived family membership and presence of cardiovascular diseases. Therefore, numbers of 53BP1 foci, telomere-associated foci (TAF) and micronuclei were measured in cultured dermal fibroblasts obtained from three age groups of donors (mean age 22, 63 and 90 years). Fibroblasts were cultured without a stressor and with 0.6 µM rotenone for 3 days. We found that 53BP1 foci and TAF were more frequently present in fibroblasts of old donors compared to middle-aged and young donors. No association between micronuclei and donor age was found. Within the fibroblasts of the middle-aged donors we did not find associations between DNA damage markers and long-lived family membership or cardiovascular disease. Results were comparable when fibroblasts were stressed in vitro with rotenone. In conclusion, we found that DNA damage foci of cultured fibroblasts are significantly associated with the chronological age, but not biological age, of the donor.

53BP1 contributes to a robust genomic stability in human fibroblasts.

Faithful repair of damaged DNA is a crucial process in maintaining cell viability and function. A multitude of factors and pathways guides this process and includes repair proteins and cell cycle checkpoint factors. Differences in the maintenance of genomic processes are one feature that may contribute to species-specific differences in lifespan. We predicted that 53BP1, a key transducer of the DNA damage response and cell cycle checkpoint control, is highly involved in maintaining genomic stability and may function differently in cells from different species. We demonstrate a difference in the levels and recruitment of 53BP1 in mouse and human cells following DNA damage. In addition, we show that unresolved DNA damage persists more in mouse cells than in human cells, as evidenced by increased numbers of micronuclei. The difference in micronuclei seems to be related to the levels of 53BP1 present in cells. Finally, we present evidence that unresolved DNA damage correlates with species lifespan. Taken together, these studies suggest a link between recruitment of 53BP1, resolution of DNA damage, and increased species lifespan.

H2O2 preconditioning modulates phase II enzymes through p38 MAPK and PI3K/Akt activation.

Ischemic preconditioning is a complex cardioprotective phenomenon that involves adaptive changes in cells and molecules and occurs in a biphasic pattern: an early phase after 1-2 h and a late phase after 12-24 h. While it is widely accepted that reactive oxygen species are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. The present study was designed to investigate the mechanisms behind H(2)O(2)-induced cardioprotection in rat neonatal cardiomyocytes. We focused on antioxidant and phase II enzymes and their modulation by protein kinase signaling pathways and nuclear-factor-E(2)-related factor-1 (Nrf1) and Nrf2. H(2)O(2) preconditioning was able to counteract oxidative stress more effectively in the late than in the early phase of adaptation. In particular, H(2)O(2) preconditioning counteracted oxidative stress-induced apoptosis by decreasing caspase-3 activity, increasing Bcl2 expression and selectively increasing the expression and activity of antioxidant and phase II enzymes through Nrf1 and Nrf2 translocation to the nucleus. The downregulation of Nrf1 and Nrf2 by small interfering RNA reduced the expression level of phase II enzymes. Specific inhibitors of phosphatidylinositol 3-kinase/Akt and p38 MAPK activation partially reduced the cardioprotection elicited by H(2)O(2) preconditioning and the induction and activity of phase II enzymes. These findings demonstrate, for the first time, a key role for Nrf1, and not only for Nrf2, in the induction of phase II enzymes triggered by H(2)O(2) preconditioning.

Metabolic stabilization of MAP kinase phosphatase-2 in senescence of human fibroblasts.

Cellular senescence is characterized by impaired cell proliferation. We have previously shown that, relative to the young counterpart, senescent WI-38 human fibroblasts display a decreased abundance of active phosphorylated ERK (p-ERK) in the nucleus. We have tested the hypothesis that this is due to elevated levels of nuclear MAP kinase phosphatase (MKP) activity in senescent cells. Our results indicate that the activity and abundance of MKP-2 is increased in senescent fibroblasts, compared to their young counterparts. Further analysis indicates that it is MKP-2 protein, but not MKP-2 mRNA level, that is increased in senescent cells. This increase is the result of the increased stability of MKP-2 protein against proteolytic degradation. The degradation of MKPs was impaired by proteasome inhibitors both in young and old WI-38 cells, indicating that proteasome activity is involved in the degradation of MKPs. Finally, our results indicate that proteasome activity, in general, is diminished in senescent fibroblasts. Taken together, these data indicate that the increased level and activity of MKP-2 in senescent WI-38 cells are the consequence of impaired proteosomal degradation, and this increase is likely to play a significant role in the decreased levels of p-ERK in the nucleus of senescent cells.

Role of the Raf/MEK/ERK and the PI3K/Akt(PKB) pathways in fibroblast senescence.

Replicative senescence is characterized by numerous phenotypic alterations including loss of proliferative capacity and numerous changes in gene expression such as impaired serum inducibility of the immediate early gene c-fos and increased expression of collagenase. Transcription of c-fos in response to mitogens depends on the activation of a multiprotein complex formed on the c-fos serum response element (SRE), which includes the transcription factors serum response factor (SRF) and ternary complex factor (TCF). TCF is activated after phosphorylation by the Extracellular signals Regulated Kinase 1 and 2 (ERK1/2), two kinases of the Raf/MEK/ERK signaling pathway. We have previously demonstrated that collagenase expression is under positive regulation by the transcription factor FKHRL1 and that this transcription factor is under negative regulation by the phosphatidylinositol 3-kinase(PI3K)/Akt(PKB) pathway. Although total activity of ERK and Akt was similar in total cell lysates from early and late passage fibroblasts our data indicate that in senescent cells neither ERK nor Akt are able to phosphorylate efficiently their nuclear targets. Our findings suggest that although they can be fully activated in the cytosol of both early and late passage cells, the Raf/MEK/ERK and the PI3K/Akt pathways, which are essential for cellular proliferation, are down regulated in the nuclei of senescent cells.

Regulation of collagenase expression during replicative senescence in human fibroblasts by Akt-forkhead signaling.

The expression of collagenase (matrix metalloproteinase 1) in human fibroblasts increases during aging both in vivo and in vitro. This age-associated increase in collagenase expression has been postulated to contribute to the age related decline in tissue function by increasing proteolysis of matrix components, but little is known regarding the regulation of collagenase expression. We examined the role that the serine/threonine kinase Akt plays in collagenase expression during in vitro senescence of WI-38 normal human lung fibroblast cells. Our results indicate that Akt-mediated signals, acting through the forkhead transcription factor FKHRL1, can regulate collagenase expression in WI-38 fibroblasts. Dominant negative forms of Akt increase collagenase promoter activity in early passage WI-38 fibroblasts, whereas an active form of Akt suppresses steady state levels of collagenase mRNA in senescent WI-38 fibroblasts. In addition, the activity of a synthetic promoter containing forkhead-specific binding sites, as measured by luciferase activity, is much higher in senescent cells compared with early passage WI-38 fibroblasts. These results indicate that members of the forkhead family of transcription factors play a role in the regulation of the collagenase promoter and that increased activity of forkhead transcription factors may underlie the increase in collagenase expression observed during replicative senescence.