Silvio De Flora

Transplacental clastogenic and epigenetic effects of gold nanoparticles in mice.

The broad application of nanotechnology in medicine, biology, and pharmacology is leading to a dramatic increase of the risk of direct contact of nanoproducts, among which gold nanoparticles (AuNP), with the human organism. The present study aimed at evaluating in vivo the genotoxicity of AuNPs with average size of 40 nm and 100 nm. A single intraperitoneal treatment of adult male and female Swiss mice (strain H) with AuNPs, at a dose of 3.3 mg/kg body weight, had no effect on the frequency of micronucleated polychromatic erythrocytes (MN PCEs) in bone marrow. Conversely, the transplacental treatment with AuNP-100 nm, but not with AuNP-40 nm, applied intraperitoneally at a dose of 3.3 mg/kg to pregnant mice on days 10, 12, 14, and 17 of gestation, resulted in a significant increase in the frequency of MN PCEs in both liver and peripheral blood of mouse fetuses. In parallel, the same treatment with AuNP-100 nm, but not with AuNP-40 nm, produced significant changes in microRNA expression. In particular, out of 1281 mouse microRNAs analyzed, 28 were dys-regulated more than two-fold and to a statistically significant extent in fetus lung, and 5 were up-regulated in fetal liver. Let-7a and miR-183 were significantly up-regulated in both organs. The data presented herein demonstrate for the first time the transplacental size-dependent clastogenic and epigenetic effects of AuNPs in mouse fetus, thus highlighting new aspects concerning the putative genotoxicity of AuNPs during a vulnerable period of life.

Cardiotoxicity of Anticancer Drugs: The Need for Cardio-Oncology and Cardio-Oncological Prevention

Due to the aging of the populations of developed countries and a common occurrence of risk factors, it is increasingly probable that a patient may have both cancer and cardiovascular disease. In addition, cytotoxic agents and targeted therapies used to treat cancer, including classic chemotherapeutic agents, monoclonal antibodies that target tyrosine kinase receptors, small molecule tyrosine kinase inhibitors, and even antiangiogenic drugs and chemoprevention agents such as cyclooxygenase-2 inhibitors, all affect the cardiovascular system. One of the reasons is that many agents reach targets in the microenvironment and do not affect only the tumor. Combination therapy often amplifies cardiotoxicity, and radiotherapy can also cause heart problems, particularly when combined with chemotherapy. In the past, cardiotoxic risk was less evident, but it is increasingly an issue, particularly with combination therapy and adjuvant therapy. Todays oncologists must be fully aware of cardiovascular risks to avoid or prevent adverse cardiovascular effects, and cardiologists must now be ready to assist oncologists by performing evaluations relevant to the choice of therapy. There is a need for cooperation between these two areas and for the development of a novel discipline, which could be termed cardio-oncology or onco-cardiology. Here, we summarize the potential cardiovascular toxicities for a range of cancer chemotherapeutic and chemopreventive agents and emphasize the importance of evaluating cardiovascular risk when patients enter into trials and the need to develop guidelines that include collateral effects on the cardiovascular system. We also discuss mechanistic pathways and describe several potential protective agents that could be administered to patients with occult or overt risk for cardiovascular complications.

Genotoxicity of chromium compounds. A review

This article reviews approximately 700 results reported in the literature with 32 chromium compounds assayed in 130 short-term tests, using different targets and/or genetic end-points. The large majority of the results obtained with Cr(VI) compounds were positive, as a function of Cr(VI) solubility and bioavailability to target cells. On the other hand, Cr(III) compounds, although even more reactive than Cr(VI) with purified nucleic acids, did not induce genotoxic effects in the majority of studies using intact cells. Coupled with the findings of metabolic studies, the large data-base generated in short-term test systems provides useful information for predicting and interpreting the peculiar patterns of Cr(VI) carcinogenicity.

Downregulation of microRNA expression in the lungs of rats exposed to cigarette smoke

Although microRNAs have been investigated extensively in cancer research, little is known regarding their response to noxious agents in apparently healthy tissues. We analyzed the expression of 484 miRNAs in the lungs of rats exposed to environmental cigarette smoke (ECS) for 28 days. ECS down‐regulated 126 miRNAs (26.0%) at least 2‐fold and 24 miRNAs more than 3‐fold. We previously demonstrated that 107 of 4858 genes (2.9%) and 50 of 518 proteins (9.7%) were up‐regulated by ECS in the same tissue, which is consistent with the role of microRNAs as negative regulators of gene expression. The most remarkably down‐regulated microRNAs belonged to the families of let‐7, miR‐10, miR‐26, miR‐30, miR‐34, miR‐99, miR‐122, miR‐123, miR‐124, miR‐125, miR‐140, miR‐145, miR‐146, miR‐191, miR‐192, miR‐219, miR‐222, and miR‐223, which regulate stress response, apoptosis, proliferation, an‐giogenesis, and expression of genes. In contrast, miR‐294, an inhibitor of transcriptional repressor genes, was up‐regulated by ECS. There was a strong parallelism in dysregulation of rodent microRNAs and their human homologues, which are often transcribed from genes localized in fragile sites deleted in lung cancer. Five ECS‐down‐regulated microRNAs are known to be affected by single nucleotide polymorphisms. Thus, changes in microRNA expression are an early event following exposure to cigarette smoke.— Izzotti, A., Calin, G. A., Arrigo, P., Steele, V. E., Croce, C. M., De Flora, S. Downregulation of microRNA expression in the lungs of rats exposed to cigarette smoke. FASEB J. 23, 806–812 (2009)

Mechanisms of inhibitors of mutagenesis and carcinogenesis

For a rational implementation of chemoprevention strategies it is essential not only to assess the efficacy and safety of putative inhibitors by using a variety of test systems but also to understand the mechanisms involved. This article proposes a detailed classification of mechanisms along with pertinent examples of agents which may be potentially exploited in the host-addressed prevention of cancer and other mutation-related diseases. The classification, presented in tabulated form, covers a variety of mechanisms interfering with different phases of mutagenesis and carcinogenesis. However, the reported sequence of events is not meant to follow a rigid scheme, and several mechanisms are reiterated throughout evolution of these processes. Similarly, a number of protective agents are shown to work through multiple and often interconnected mechanisms.

‘Angioprevention’: angiogenesis is a common and key target for cancer chemopreventive agents

The potential to block tumor growth by inhibition of the neoangiogenic process represents an intriguing approach to the treatment of solid tumors. The high proliferation rate in the tumor deprived of proper vascularization would be balanced by cell death due to lack of diffusion of nutrients and oxygen. Matrix metalloproteinases (MMPs), angiogenic growth factors, and their receptors are the main targets of an increasing number of clinical trials approved to test the tolerance and therapeutic efficacy of antiangiogenic agents. We observed that a series of substances proposed as possible cancer chemopreventive agents show antiangiogenic properties when tested in in vitro and in vivo angiogenesis models. We demonstrated that N‐acetyl‐L‐cysteine is able to reduce the invasive and metastatic potential of melanoma cells, and to inhibit endothelial cell invasion by direct inhibition of MMP activity. We also showed that epigallocatechin gallate (EGCG), a flavonoid from green tea that possesses chemopreventive activity in experimental and epidemi‐ological studies, is a potent inhibitor of MMP‐2 and MMP‐9. Angiogenesis has also been demonstrated to be a target for nonsteroidal anti‐inflammatory drug che‐mopreventive activity. Based on these data, we hypothesize that other chemopreventive agents, including natural or synthetic retinoids, steroid hormone antagonists, peroxisome proliferator‐activated receptor γ li‐gands, vitamin D, and protease inhibitors, might have antiangiogenesis as an important mechanism of action, a novel concept we will term ‘angioprevention’. We analyze the mechanisms on how and why chemopreven‐tive agents could exert antiangiogenic effects aimed at controlling tumor growth, and their potential use in the clinic.—Tosetti, F., Ferrari, N., De Flora, S., Albini, A. ‘Angioprevention’: angiogenesis is a common and key target for cancer chemopreventive agents. FASEB J. 16, 2–14 (2002)

Mechanisms of inhibitors of mutagenesis and carcinogenesis. Classification and overview

The mechanisms of action of inhibitors of mutagenesis and carcinogenesis are reviewed in the light of our present knowledge. The identified mechanisms are classified into several categories and sub-categories, depending on the stage of intervention in the mutagenesis and carcinogenesis processes, and on the patterns of modulation of the host defense devices. Although a number of the known mechanisms fit into the proposed scheme, the available information on these problems is still fragmentary, and often inhibitors act through multiple mechanisms or can interact with other inhibitors. Moreover, due to the double-edged nature of many protective factors of the organism, and to the wide array of biological properties displayed by several inhibitors, the beneficial effects are in many instances counter-balanced by adverse reactions. Nevertheless, the present data-base on mechanisms of inhibitors, which is expected to grow rapidly in the near future, provides an extremely useful scientific premise for the primary prevention of mutation-related diseases. In this prospect, the elucidation of the underlying mechanisms complements the results emerging from the monitoring of protective end-points in mutagenicity and carcinogenicity test systems.

N-acetyl-l-cysteine

The most commonly used chemopreventive agents in the prevention of oral leukoplakia, head and neck cancer, and lung cancer are β‐carotene, vitamin A, and other retinoids. One of the few chemopreventive agents not in this group and presently being used in a clinical trial is N‐acetyl‐l‐cysteine (NAC). NAC, an antioxidant, is used in EUROSCAN, a European Organization of Research and Treatment of Cancer (EORTC) chemoprevention study in curatively treated patients with oral, laryngeal, or lung cancer. The rationale for choosing NAC is based on a variety of experimental data showing its ability to exert protective effects, including extracellular inhibition of mutagenic agents from exogenous and endogenous sources, inhibition of genotoxicity of reactive oxygen species, modulation of metabolism coordinated with blocking of reactive metabolites, protection of DNA and nuclear enzymes, and prevention of the formation of carcinogen–DNA adducts. NAC has also demonstrated an effect on mutagen‐induced chromosomal sensitivity assays, and on anticarcinogenicity in experimental animal models. In addition, preliminary data from EUROSCAN show good compliance in treated patients and a low frequency of side effects.

Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair test

Compounds of various chemical classes were comparatively assayed in the Ames reversion test with his- S. typhimurium strains TA1535, TA157 , TA1538, TA98, TA100, and, in part, TA97 , and in a DNA-repair test with trp- E. coli strains WP2 (repair-proficient), WP67 (uvrA- polA-) and CM871 (uvrA- recA- lexA-). A liquid micromethod procedure for the assessment of the minimal inhibitory concentration (MIC) of test compounds, using the same reagents as the Ames test, was set up and calibrated in its technical details. Other techniques (spot test and treat-and-plate method) were applied to a number of compounds in order to obtain more complete information on their DNA-damaging activity in E. coli. From a qualitative standpoint, the results obtained in the reversion test and in the DNA-repair test (liquid micromethod) were overlapping for 96 (59 positive and 37 negative) out of 135 compounds (71.1%). There was disagreement for 39 compounds (28.9%), 9 of which were positive only in the reversion test (8 requiring metabolic activation and 5 genotoxic in the treat-and-plate method). 30 compounds were positive only in the lethality test, showing a direct DNA-damaging activity, which in half of the cases was completely eliminated by S9 mix. Although the experimental protocol intentionally included several compounds already reported as nonmutagenic carcinogens or as noncarcinogenic mutagens, the overall accuracy was 64.5% for the reversion test and 72.4% for the DNA-repair test, as evaluated for 75 compounds classified according to their carcinogenic activity. Quantitation of results was obtained in the Ames test by relating the net number of revertants to nmoles of compound and in the DNA-repair test by means of a formula relating the difference and ratio of MICs in repair-proficient and -deficient bacteria to nmoles of compound. Following these criteria, the genotoxic potency varied over a 4.5 X 10(7)-fold range among compounds positive in the reversion test and over a 6 X 10(9)-fold range among compounds damaging E. coli DNA. The genotoxic potencies in the two bacterial systems were correlated within the majority of the chemical classes under scrutiny.

DNA adducts and chronic degenerative diseases. Pathogenetic relevance and implications in preventive medicine

Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques. In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type II-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type II-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/ dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.