HPV Infection Leaves a DNA Methylation Signature in Oropharyngeal Cancer Affecting Both Coding Genes and Transposable Elements.

HPV oncoproteins can modulate DNMT1 expression and activity, and previous studies have reported both gene-specific and global DNA methylation alterations according to HPV status in head and neck cancer. However, validation of these findings and a more detailed analysis of the transposable elements (TEs) are still missing. Here we performed pyrosequencing to evaluate a 5-CpG methylation signature and Line1 methylation in an oropharyngeal squamous cell carcinoma (OPSCC) cohort. We further evaluated the methylation levels of the TEs, their correlation with gene expression and their impact on overall survival (OS) using the TCGA cohort. In our dataset, the 5-CpG signature distinguished HPV-positive and HPV-negative OPSCC with 66.67% sensitivity and 84.33% specificity. Line1 methylation levels were higher in HPV-positive cases. In the TCGA cohort, Line1, Alu and long terminal repeats (LTRs) showed hypermethylation in a frequency of 60.5%, 58.9% and 92.3%, respectively. ZNF541 and CCNL1 higher expression was observed in HPV-positive OPSCC, correlated with lower methylation levels of promoter-associated Alu and LTR, respectively, and independently associated with better OS. Based on our findings, we may conclude that a 5-CpG methylation signature can discriminate OPSCC according to HPV status with high accuracy and TEs are differentially methylated and may regulate gene expression in HPV-positive OPSCC.

Co-expression network analysis identifies novel molecular pathways associated with cadmium and pyriproxyfen testicular toxicity in Gammarus fossarum.

Omics approaches are continuously providing new clues on the mechanisms of action of contaminants in species of environmental relevance, contributing to the emergence of molecular ecotoxicology. Co-expression network approaches represent a suitable methodological framework for studying the rich content of omics datasets. This study aimed to find evidence of key pathways and proteins related to the testicular toxicity in the sentinel crustacean species Gammarus fossarum exposed to endocrine disruptors using a weighted protein co-expression network analysis. From a shotgun proteomics dataset of male gonads of G. fossarum organisms exposed to cadmium (Cd), pyriproxyfen (Pyr) and methoxyfenozide (Met) in laboratory conditions, four distinct modules were identified as significantly correlated to contaminants' exposure. Protein set enrichment analysis identified modules involved in cytoskeleton organization and oxidative stress response associated with the Cd exposure. The module associated with Pyr exposure was associated with endoplasmic reticulum stress (ER) response, and the module correlated with Met exposure was characterized by a significant proportion of amphipod-restricted proteins whose functions are still not characterized. Our results show that co-expression networks are efficient and adapted tools to identify new potential mode of actions from environmental sentinel species, such as G. fossarum, using a proteogenomic approach, even without an annotated genome.

Gate-Controlled Suspended Titanium Nanobridge Supercurrent Transistor.

Under standard conditions, the electrostatic field-effect is negligible in conventional metals and was expected to be completely ineffective also in superconducting metals. This common belief was recently put under question by a family of experiments that displayed full gate-voltage-induced suppression of critical current in superconducting all-metallic gated nanotransistors. To date, the microscopic origin of this phenomenon is under debate, and trivial explanations based on heating effects given by the negligible electron leakage from the gates should be excluded. Here, we demonstrate the control of the supercurrent in fully suspended superconducting nanobridges. Our advanced nanofabrication methods allow us to build suspended superconducting Ti-based supercurrent transistors which show ambipolar and monotonic full suppression of the critical current for gate voltages of VGC ≃ 18 V and for temperatures up to ∼80% of the critical temperature. The suspended device architecture minimizes the electron-phonon interaction between the superconducting nanobridge and the substrate, and therefore, it rules out any possible contribution stemming from charge injection into the insulating substrate. Besides, our finite element method simulations of vacuum electron tunneling from the gate to the bridge and thermal considerations rule out the cold-electron field emission as a possible driving mechanism for the observed phenomenology. Our findings promise a better understanding of the field effect in superconducting metals.

A transcriptomic analysis of black cohosh: Actein alters cholesterol biosynthesis pathways and synergizes with simvastatin.

Previous studies indicate that the herb black cohosh (Actaea racemosa L.) and the triterpene glycoside actein inhibit the growth of human breast cancer cells and activate stress-associated responses. This study assessed the transcriptomic effects of black cohosh and actein on rat liver tissue, using Ingenuity and ToxFX analyses. Sprague-Dawley rats were treated with an extract of black cohosh enriched in triterpene glycosides (27%) for 24 h or actein for 6 and 24 h, at 35.7 mg/kg, and liver tissue collected for gene expression analysis. Ingenuity analysis indicates the top canonical pathways are, for black cohosh, RAR Activation, and, for actein, Superpathway of Cholesterol Biosynthesis, at 24 h. Actein alters the expression of cholesterol biosynthetic genes, but does not inhibit HMG-CoA reductase activity. Black cohosh and actein inhibited the growth of human breast and colon cancer cells and synergized with the statin simvastatin. Combinations of black cohosh with certain classes of statins could enhance their activity, as well as toxic, such as inflammatory liver, side effects. Transcriptomic analysis indicates black cohosh and actein warrant further study to prevent and treat cancer and lipid disorders. This study lays the basis for an approach to characterize the mode of action and toxicity of herbal medicines.

Antiproliferative Effects of Epigenetic Modifier Drugs Through E-cadherin Up-regulation in Liver Cancer Cell Lines.

INTRODUCTION AND AIM: Epigenetic alterations play an essential role in cancer onset and progression, thus studies of drugs targeting the epigenetic machinery are a principal concern for cancer treatment. Here, we evaluated the potential of the combination of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5aza-dC) and the pan-deacetylase inhibitor Trichostatin A (TSA), at low cytotoxic concentrations, to modulate the canonical Wnt/ß-catenin pathway in liver cancer cells. MATERIAL AND METHODS: Pyrosequencing was used for DNA methylation analyses of LINE-1 sequences and the Wnt/ß-catenin pathway antagonist DKK3, SFRP1, WIF1 and CDH1. qRT-PCR was employed to verify the expression of the antagonist. Pathway regulation were evaluated looking at the expression of ß-catenin and E-cadherin by confocal microscopy and the antitumoral effects of the drugs was studied by wound healing and clonogenic assays. RESULTS: Our result suggest that 5aza-dC and TSA treatments were enough to induce a significant expression of the pathway antagonists, decrease of ß-catenin protein levels, re-localization of the protein to the plasma membrane, and pathway transcriptional activity reduction. These important effects exerted an antitumoral outcome shown by the reduction of the migration and clonogenic capabilities of the cells. CONCLUSION: We were able to demonstrate Wnt/ ß-catenin pathway modulation through E-cadherin up-regulation induced by 5aza-dC and TSA treatments, under an activation-pathway background, like CTNNB1 and TP53 mutations. These findings provide evidences of the potential effect of epigenetic modifier drugs for liver cancer treatment. However, further research needs to be conducted, to determine the in vivo potential of this treatment regimen for the management of liver cancer.

Identification of novel long non-coding RNAs deregulated in hepatocellular carcinoma using RNA-sequencing.

Functional characterization of long non-coding RNAs (lncRNAs) and their pathological relevance is still a challenging task. Abnormal expression of a few long non-coding RNAs have been found associated with hepatocellular carcinoma, with potential implications to both improve our understanding of molecular mechanism of liver carcinogenesis and to discover biomarkers for early diagnosis or therapy. However, the understanding of the global role of lncRNAs during HCC development is still in its infancy. In this study, we produced RNA-Seq data from 23 liver tissues (controls, cirrhotic and HCCs) and applied statistical and gene network analysis approaches to identify and characterize expressed lncRNAs. We detected 5,525 lncRNAs across different tissue types and identified 57 differentially expressed lncRNAs in HCC compared with adjacent non-tumour tissues using stringent criteria (FDR<0.05, Fold Change>2). Using weighted gene co-expression network analysis (WGCNA), we found that differentially expressed lncRNAs are co-expressed with genes involved in cell cycle regulation, TGF-ß signalling and liver metabolism. Furthermore, we found that more than 20% of differentially expressed lncRNAs are associated to actively transcribed enhancers and that the co-expression patterns with their closest genes change dramatically during HCC development. Our study provides the most comprehensive compendium of lncRNAs expressed in HCC, as well as in control or cirrhotic livers. Our results identified both known oncogenic lncRNAs (such as H19 and CRNDE) and novel lncRNAs involved in cell cycle deregulation and liver metabolism deficits occurring during HCC development.

Ischemic Postconditioning of the Liver Graft in Adult Liver Transplantation.

BACKGROUND: Ischemia-reperfusion (I/R) injury is the main cause of graft failure in liver transplantation (LT). Ischemic postconditioning (IPo) has shown to be beneficial against I/R injury. Our objective was to compare the results of LT with or without IPo. METHODS: One hundred patients undergoing LT alternatively received IPo or not. At the time of arterial reperfusion, IPo consisted of three 1-minute arterial occlusions, interspersed with 1-minute reperfusion pauses. The primary endpoint was postoperative aspartate aminotransferase (AST) peak value; early graft dysfunction and histological I/R injury were secondary endpoints. RESULTS: Median postoperative AST peak values was similar in both groups (426 vs 463 IU/L, P = 0.21); no difference was found in other postoperative liver function tests. In the IPo group, fewer grafts presented severe histological I/R injury (12% vs 28%; P = 0.029). Ischemic postconditioning did not induce changes in cellular apoptosis but triggered autophagy in periportal areas. Independent predictors of severe I/R injury were IPo (odds ratio, 0.20; P = 0.008) and arterial warm ischemia duration (odds ratio, 1.05; P = 0.008). Early graft dysfunction rate was similar in both groups (20% versus 26%, P = 0.47) and was associated with severe histological I/R injury and longer cold ischemia. Morbidity, mortality, and 1-year graft and patient survival were similar in both groups. CONCLUSIONS: Ischemic postconditioning did not influence postoperative AST peak values or other liver function tests. However, our results showed a better tolerance to I/R injury on histological findings of grafts receiving IPo. Future studies are necessary to optimize the IPo protocol in LT, to clarify its clinical impact, and to deepen the molecular understanding.

Aberrant DNA methylation of imprinted loci in hepatocellular carcinoma and after in vitro exposure to common risk factors.

BACKGROUND: Hepatocellular carcinoma (HCC) is among the most frequent human malignancies and a major cause of cancer-related death worldwide. It is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be one of the molecular mechanisms leading to hepatocarcinogenesis. Previous studies from our group revealed that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression. Imprinted genes are mono-allelically expressed in a parent-of-origin-dependent manner and have been suggested to be more susceptible to deregulation in cancer. To test this notion, we performed a targeted analysis of DNA methylation in known imprinted genes, using HCC samples and in vitro models of carcinogenic exposure. RESULTS: Analysis of HCC DNA methylation in two independent datasets showed that differentially methylated loci are significantly enriched in imprinted genes. Most of the promoters of imprinted genes were found hypomethylated in HCC tumors compared to surrounding tissues, contrasting with the frequent promoter hypermethylation observed in tumors. We next investigated the status of methylation of the imprinting control region (ICR) of different imprinted clusters and found that the 15q11-13 ICR was significantly hypomethylated in tumors relative to their surrounding tissues. In addition, expression of imprinted genes within this cluster was frequently deregulated in a gene-specific manner, suggesting distinct mechanisms of regulation in this region. Finally, primary human hepatocytes and hepatocyte-like HepaRG cells displayed higher methylation variability in certain imprinted loci after natural hepatitis B virus (HBV) infection and after lipid accumulation, respectively. CONCLUSION: The methylation status of a large panel of imprinted genes was found deregulated in HCC, suggesting a major role of this mechanism during hepatocarcinogenesis. In vitro models support the hypothesis of imprinted gene methylation as a potential marker of environmental exposures.

Glycogen synthase kinase 3-mediated voltage-dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation.

UNLABELLED: Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early mitochondrial impairment and late hepatocyte cell death. However, mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high-fat diet, and in vitro models of lipotoxicity, we show that outer mitochondrial membrane permeability is altered and identified a posttranslational modification of voltage-dependent anion channel (VDAC), a membrane channel and NADH oxidase, as a cause of early mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. CONCLUSION: VDAC acts as an early sensor of lipid toxicity and its GSK3-mediated phosphorylation status controls outer mitochondrial membrane permeabilization in hepatosteatosis.

Non-alcoholic steatohepatitis: new insights from OMICS studies.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver pathology characterized by fat accumulation in a context of metabolic syndrome or insulin resistance. It can be associated with obesity, diabetes, hyperinsulinemia, dyslipidemia as well as hypertension. NAFLD consists of a large spectrum of hepatic lesions including benign steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis or hepatocellular carcinoma. Upon chronic stress, NASH would occur via at least "two-hits" process involving modulation of a high number of genes and proteins. Firstly, the accumulation of fat, either due to the increased inflow of free fatty acids or de novo lipogenesis, leads to steatosis. Secondly, when adaptive mechanisms for stress tolerance are overwhelmed, lipotoxicity and chronic inflammation trigger major hepatic damages, mainly via oxidative and inflammatory stress, lipid peroxidation and cell death. As a consequence, all these processes concur to favor steatohepatitis, fibrosis and cancer. Recently, the elucidation of physiopathological signaling cascades controlling NAFLD and NASH benefited from large-scale studies, namely the omics, such as transcriptomics, genomics, proteomics, and lipidomics. The advent of lipidomics would allow shedding light upon the respective roles of triglyceride and fatty acid metabolites in the lipotoxic liver injury hypothesis for the pathogenesis of NASH. In this review, the contribution of the omics to the understanding of the molecular basis of NASH is discussed that could offer perspectives for novel biomarkers discovery.

Pharmacological mechanisms of black cohosh in Sprague-Dawley rats.

BACKGROUND: Studies indicate that extracts and purified components from black cohosh inhibit the growth of human breast cancer cells, but the molecular targets and signaling pathways have not yet been defined. PURPOSE: This study examines the pharmacological mechanisms and toxicological effects in the short term of the herb black cohosh on female Sprague-Dawley rats. MATERIALS AND METHODS: To assess effects on gene activity and lipid content, we treated female Sprague-Dawley rats with an extract of black cohosh enriched in triterpene glycosides (27%) at 35.7 or 0mg/kg. Four animals for each group were sacrificed at 1, 6 and 24h after treatment; liver tissue and serum samples were obtained for gene expression and lipid analysis. RESULTS: Microarray analysis of rat liver tissue indicated that black cohosh markedly downregulated mitochondrial oxidative phosphorylation genes. Phospholipid biosynthesis and remodeling, PI3-Kinase and sphingosine signaling were upregulated, driven largely by an upregulation of several isoforms of phospholipase C. Hierarchical clustering indicated that black cohosh clustered with antiproliferative compounds, specifically tubulin binding vinca alkaloids and DNA alkylators. In support of this, black cohosh repressed the expression of cyclin D1 and ID3, and inhibited the proliferation of HepG2, p53 positive, liver cancer cells. Black cohosh reduced the level of free fatty acids at 6 and 24h and triglycerides at 6h in the serum, but increased the free fatty acid and triglyceride content of the treated livers at 24h. CONCLUSION: Our results suggest that black cohosh warrants further study for breast cancer prevention and therapy.

Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2.

A high resistance and heterogeneous response to conventional anti-cancer chemotherapies characterize malignant cutaneous melanoma, the most aggressive and deadly form of skin cancer. Withaferin A (WFA), a withanolide derived from the medicinal plant Withania somnifera, has been reported for its anti-tumorigenic activity against various cancer cells. For the first time, we examined the death-inducing potential of WFA against a panel of four different human melanoma cells and investigated the cellular mechanisms involved. WFA induces apoptotic cell death with various IC50 ranging from 1.8 to 6.1 µM. The susceptibility of cells toward WFA-induced apoptosis correlated with low Bcl-2/Bax and Bcl-2/Bim ratios. In all cell lines, the apoptotic process triggered by WFA involves the mitochondrial pathway and was associated with Bcl-2 down regulation, Bax mitochondrial translocation, cytochrome c release into the cytosol, transmembrane potential (ΔΨm) dissipation, caspase 9 and caspase 3 activation and DNA fragmentation. WFA cytotoxicity requires early reactive oxygen species (ROS) production and glutathione depletion, the inhibition of ROS increase by the antioxidant N-acetylcysteine resulting in complete suppression of mitochondrial and nuclear events. Altogether, these results support the therapeutic potential of WFA against human melanoma.

Autophagy is induced by ischemic preconditioning in human livers formerly treated by chemotherapy to limit necrosis.

The effectiveness of ischemic preconditioning (IP) against hepatic ischemia/reperfusion injury during human liver surgery is linked to decreased apoptotic cell death as well as preservation of the ATP content in liver tissue. Overproduction of Bcl-2 is reported in preconditioned organs. In human liver biopsies exhibiting steatosis and/or vascular injuries (mainly peliosis) induced by chemotherapy, we find that the expression of Bcl-2 in centrolobular and peliotic areas colocalizes with the autophagy protein Beclin 1 in IP livers. Increased expression of phosphorylated Bcl-2 in preconditioned livers is associated with decreased immunoprecipitation of Beclin 1 and increased expression of LC3-II. The increased number of autophagic vacuoles seen by electron microscopy confirmed that IP could trigger autophagy in chemotherapy-injured livers, probably to reduce the pro-inflammatory necrotic cell death of hepatocytes or endothelial cells and to increase ATP levels. Indeed, necrosis is less frequent (p = 0.04) in IP livers than in the others although no change in apoptosis as assessed by TUNEL assay or caspase-3, -8 and -9 expressions is observed. In conclusion, Bcl-2 and Beclin 1 could be major targets in the regulation of cell death during ischemia/reperfusion injury modulating autophagy to switch on/off necrosis and/or apoptosis.

Concurrent induction of necrosis, apoptosis, and autophagy in ischemic preconditioned human livers formerly treated by chemotherapy.

BACKGROUND/AIMS: Liver pathology induced by chemotherapy (steatosis or vascular injury) is known to increase the liver's sensitivity to ischemia/ reperfusion (I/R) injury, thereby increasing morbidity and mortality after liver resection. Our aim was to assess whether ischemic preconditioning (IP) reduces I/R injury to livers with chemotherapy-induced pathology. METHODS: We analyzed a series of livers from patients treated with chemotherapy for colorectal cancer who underwent IP (n=30) or not (n=31) before hepatectomy. All but one of the livers exhibited chemotherapy-induced steatosis and/ or peliosis before the I/R insult. RESULTS: Necrosis was less frequent (p=0.038) in livers with IP than in the others. IP had no influence on apoptosis as assessed by terminal transferase uridyl nick-end labeling (TUNEL) assay or caspase-3, -8 and -9 expression. IP induced a twofold increase in B-cell leukemia/ lymphoma 2 (Bcl-2; p<0.05), which was localized to hepatocytes of centrolobular and peliotic areas and colocalized with the autophagy protein beclin-1 in livers with IP, suggesting their coordinated role in autophagy. Increased expression of the phosphorylated Bcl-2 was observed in preconditioned livers and was associated with a decreased immunoprecipitation of beclin-1 and the increased expression of light chain 3 type II (LC3-II). The increased number of autophagic vacuoles seen by electron microscopy confirmed an association of autophagy in chemotherapy-injured livers following IP. However, the differences in protein expression were not reflected in postresection liver-injury tests or measure of patient morbidity. CONCLUSIONS: IP is associated with a reduction in necrosis of hepatocytes already damaged by chemotherapy and an activation of autophagy. Bcl-2 and beclin-1 could be major targets in the regulation of cell death during I/R injury.

Actein activates stress- and statin-associated responses and is bioavailable in Sprague-Dawley rats.

The purpose of this study was to assess in rats the pharmacological parameters and effects on gene expression in the liver of the triterpene glycoside actein. Actein, an active component from the herb black cohosh, has been shown to inhibit the proliferation of human breast cancer cells. To conduct our assessment, we determined the molecular effects of actein on livers from Sprague-Dawley rats treated with actein at 35.7 mg/kg for 6 and 24 h. Chemogenomic analyses indicated that actein elicited stress and statin-associated responses in the liver; actein altered expression of cholesterol and fatty acid biosynthetic genes, p53 pathway genes, CCND1 and ID3. Real-time RT-PCR validated that actein induced three time-dependent patterns of gene expression in the liver: (i) a decrease followed by a significant increase of HMGCS1, HMGCR, HSD17B7, NQO1, S100A9; (ii) a progressive increase of BZRP and CYP7A1 and (iii) a significant increase followed by a decrease of CCND1 and ID3. Consistent with actein's statin- and stress-associated responses, actein reduced free fatty acid and cholesterol content in the liver by 0.6-fold at 24 h and inhibited the growth of human HepG2 liver cancer cells. To determine the bioavailability of actein, we collected serum samples for pharmacokinetic analysis at various times up to 24 h. The serum level of actein peaked at 2.4 microg/mL at 6 h. Actein's ability to alter pathways involved in lipid disorders and carcinogenesis may make it a new agent for preventing and treating these major disorders.

Consequences of exposure to carcinogens beginning during developmental life.

The increased incidence of cancer over the last 50-60 years may be largely attributed to two factors: the ageing of the population and the diffusion of agents and situations presenting carcinogenic risks. Today, we have entered into a new era in which populations are ever-increasingly exposed to diffuse carcinogenic risks, present not only in the occupational, but also in the general environment. We must now also consider an additional factor in the carcinogenic process, that is, the age in which exposure to carcinogenic risks begins. Apart from the paradigmatic cases of diethylstilboestrol and ionizing radiation, the available epidemiological data concerning the adult consequences of developmental exposure to carcinogens is very limited. However, important data have been provided by long-term experimental carcinogenicity bioassays conducted using rodents. This paper reports a selection of studies conducted in the laboratories of the Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation in which exposure to the chemical agents vinyl acetate monomer, ethyl alcohol and aspartame was started during developmental life and continued into adulthood. The results of these studies provide supporting evidence that lifespan exposure to carcinogenic agents beginning during developmental life produces an overall increase in the carcinogenic effects observed. Moreover, when comparing prenatal and postnatal exposure, the data demonstrate that the development of cancers may appear earlier in life.

Life-span exposure to low doses of aspartame beginning during prenatal life increases cancer effects in rats.

BACKGROUND: In a previous study conducted at the Cesare Maltoni Cancer Research Center of the European Ramazzini Foundation (CMCRC/ERF), we demonstrated for the first time that aspartame (APM) is a multipotent carcinogenic agent when various doses are administered with feed to Sprague-Dawley rats from 8 weeks of age throughout the life span. OBJECTIVE: The aim of this second study is to better quantify the carcinogenic risk of APM, beginning treatment during fetal life. METHODS: We studied groups of 70-95 male and female Sprague-Dawley rats administered APM (2,000, 400, or 0 ppm) with feed from the 12th day of fetal life until natural death. RESULTS: Our results show a) a significant dose-related increase of malignant tumor-bearing animals in males (p < 0.01), particularly in the group treated with 2,000 ppm APM (p < 0.01); b) a significant increase in incidence of lymphomas/leukemias in males treated with 2,000 ppm (p < 0.05) and a significant dose-related increase in incidence of lymphomas/leukemias in females (p < 0.01), particularly in the 2,000-ppm group (p < 0.01); and c) a significant dose-related increase in incidence of mammary cancer in females (p < 0.05), particularly in the 2,000-ppm group (p < 0.05). CONCLUSIONS: The results of this carcinogenicity bioassay confirm and reinforce the first experimental demonstration of APM's multipotential carcinogenicity at a dose level close to the acceptable daily intake for humans. Furthermore, the study demonstrates that when life-span exposure to APM begins during fetal life, its carcinogenic effects are increased.