m6A modification mediates SLC3A2/SLC7A5 translation in 3-methylcholanthrene-induced uroepithelial transformation.

3-Methylcholanthracene (3-MC) is one of the most carcinogenic polycyclic aromatic hydrocarbons (PAHs). Long-term exposure to PAHs has been thought of as an important factor in urothelial tumorigenesis. N6-methyladenosine (m6A) exists widely in eukaryotic organisms and regulates the expression level of specific genes by regulating mRNA stability, translation efficiency, and nuclear export efficiency. Currently, the potential molecular mechanisms that regulate m6A modification for 3-MC carcinogenesis remain unclear. Here, we profiled mRNA, m6A, translation and protein level using "-omics" methodologies, including transcriptomes, m6A profile, translatomes, and proteomics in 3-MC-transformed urothelial cells and control cells. The key molecules SLC3A2/SLC7A5 were screened and identified in 3-MC-induced uroepithelial transformation. Moreover, SLC7A5/SLC3A2 promoted uroepithelial cells malignant phenotype in vitro and in vivo. Mechanically, METTL3 and ALKBH5 mediated m6A modification of SLC3A2/SLC7A5 mRNA in 3-MC-induced uroepithelial transformation by upregulating the translation of SLC3A2/SLC7A5. Furthermore, programmable m6A modification of SLC3A2/SLC7A5 mRNA affected the expression of its proteins. Taken together, our results revealed that the m6A modification-mediated SLC3A2/SLC7A5 translation promoted 3-MC-induced uroepithelial transformation, suggesting that targeting m6A modification of SLC3A2/SLC7A5 may be a potential therapeutic strategy for bladder cancer related to PAHs.

Aryl Hydrocarbon Receptor Directly Regulates VTCN1 Gene Expression in MCF-7 Cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.

Parental exposure 3-methylcholanthrene disturbed the enterohepatic circulation in F1 generation of mice.

3-methylcholanthrene (3 MC) is an environmental compound belonging to the PAHs and is reportedly thought to be a risk factor for the prevalence of hepatic function disorder. Here, a dose of 0.5 mg/kg of 3 MC was given to 4-week-old male and female mice (F0) in their diet for 6 weeks. After exposure, then the mice were mated between different groups. The first filial (F1) generation offspring of exposed or unexposed parental mice were sacrificed at the age of 5 weeks (F1-5 W), and the potential effects on the F0 and F1 offspring were evaluated. The results showed that the total bile acids (TBAs) in the serum and feces in F0 females and female F1-5 W individuals born from female mice exposed to 3 MC decreased, while the TBAs in the liver increased. The transcriptional levels of major genes participating in synthesis, regulation, transportation and apical uptake was also altered correspondingly. In addition, the transcription of some genes related to inflammation was enhanced in these mice. Further investigation revealed that in addition to distinct changes in the mucus secretion, tight junction proteins and ion transport were induced, and antimicrobial peptides were also disrupted in the intestine of F0 mice and F1-5 W female offspring of maternal mice exposed to 3 MC. Our results suggested that exposure to 3 MC, but not male exposure, had the potential to interfere with BAs metabolism, affecting gut barrier function. Females were more seriously affected than males.

Metformin alters therapeutic effects in the BALB/c tumor therapy model.

BACKGROUND: Despite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin. METHODS: Cytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting. RESULTS: Initial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells. CONCLUSION: In conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy.

Two-stage 3-methylcholanthrene and butylated hydroxytoluene-induced lung carcinogenesis in mice.

Lung cancer is one of the deadliest types of cancer and as such requires disease models that are useful for identification of novel pathways for biomarkers as well as to test therapeutic agents. Adenocarcinoma (ADC), the most prevalent type of lung cancer, is a subtype of non-small cell lung carcinoma (NSCLC) and a disease driven mainly by smoking. However, it is also the most common subtype of lung cancer found in non-smokers with environmental exposures. Chemically driven models of lung cancer, also called primary models of lung cancer, are important because they do not overexpress or delete oncogenes or tumor suppressor genes, respectively, to increase oncogenesis. Instead these models test tumor development without forcing a specific pathway (i.e., Kras). The primary focus of this chapter is to discuss a well-established 2-stage mouse model of lung adenocarcinomas. The initiator (3-methylcholanthrene, MCA) does not elicit many, if any, tumors if not followed by exposure to the tumor promoter (butylated hydroxytoluene, BHT). In sensitive strains, such as A/J, FVB, and BALB, significantly greater numbers of tumors develop following the MCA/BHT protocol compared to MCA alone. BHT does not elicit tumors on its own; it is a non-genotoxic carcinogen and promoter. In these sensitive strains, promotion is also associated with inflammation characterized by infiltrating macrophages, lymphocytes, and neutrophils, and other inflammatory cell types in addition to increases in total protein content reflective of lung hyperpermeability. This 2-stage model is a useful tool to identify unique promotion specific events to then test in future intervention studies.

Using methylcholanthrene-induced fibrosarcomas to study tumor immunology.

Mouse models of cancer are essential in furthering our understanding both of the mechanisms that drive tumor development and the immune response that develops in parallel, and also in providing a platform for testing novel anti-cancer therapies. The majority of solid tumor models available rely on the injection of existing cancer cell lines into naïve hosts which, while providing quick and reproducible model systems, typically lack the development of a tumor microenvironment that recapitulates those seen in human cancers. Administration of the carcinogen 3-methylcholanthrene (MCA), allows tumors to develop in situ, forming a tumor microenvironment with an established stroma and vasculature. This article provides a detailed set of protocols for the administration of MCA into mice and the subsequent monitoring of tumors. Protocols are also provided for some of the routinely used downstream applications that can be used for MCA tumors.

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

Late effects of cutaneous 3-methylcholanthrene exposure on DNA damage-related pleiotropic growth factors and oxidative stress markers in mice.

BACKGROUND: Skin is the body's first defence against direct exposure to variety of chemicals. Polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (3-MC) are common in polluted urban air and have a potential of producing harmful effects. Moreover, their late effects can occur months or years after exposure. OBJECTIVES: We aimed to investigate the long-term effects of 3-MC induced dermal toxicity on the expression of markers of apoptosis, pleiotropic cytokines, and oxidative stress and to determine the protective effect of cisplatin. METHODS: Groups were designed as control (group 1), 3-MC applied (group 2) and 3-MC+cisplatin applied mice (group 3). Cutaneous expressions of TGFß, PDGFA, PDGFC, bFGF, PDGFRα, USP28, and Ki67 were evaluated with qPCR. Total oxidant (TOS), total antioxidant (TAS) and oxidative stress index (OSI) values were determined in liver and kidney tissues. RESULTS: The expression levels of TGFß, PDGFRα, USP-28, Ki67, and PDGFA were decreased significantly in MC applied groups. Renal TAS levels were significantly lower in group-3. Liver and kidney OSI values were increased in both groups 2 and 3. CONCLUSION: The results indicated that low dose 3-MC caused oxidative stress and downregulated apoptotic and cytokine markers in the long term and cisplatin had no ameliorative effects on this degeneration processes (Tab. 3, Fig. 3, Ref. 32). Text in PDF www.elis.sk.

3-Methylcholanthrene impacts on the female germ cells of rats without causing systemic toxicity.

We have previously shown that daily exposure to the environmental pollutant 3-methylcholanthrene (3MC) alters the ovarian function by affecting follicle growth and ovulation. To extend our findings, the aims of this work were to study the effects of daily and non-daily exposure to 3MC on oocyte morphology and integrity and the meiosis process. To this end, immature female rats were daily (0.1-1.0 mg/kg) and non-daily (0.1 mg/kg, three times a week) exposed to 3MC and/or α-naphthoflavone (αNF) (80 mg/kg) for 19 and 20 days, respectively. The latter was used to study its ability to prevent the 3MC action. Follicular growth was examined by histology, apoptosis by in situ cell death detection, oocyte integrity by morphological parameters and fluorescent dyes, and the meiotic spindle by immunostaining. Compared with controls (C), and in a dose-dependent manner, all 3MC-treated rats showed i) increased presence of apoptotic cells in antral follicles and decreased percentage of healthy oocytes, ii) increased oocyte area, perimeter and perivitelline space and decreased thickness of the zona pellucida, and ii) increased percentage of oocytes with abnormal meiotic spindle. In addition, the non-daily dose of 3MC caused DNA damage in oocytes, but not in blood or bone marrow cells. All 3MC-induced changes were prevented with the co-treatment with αNF. These results suggest that low doses of 3MC severely disrupt the ovarian function and that germ cells seem to be more sensitive to this environmental pollutant than other cells such as peripheral blood and bone marrow cells.

3-Methylcholanthrene Induces Chylous Ascites in TCDD-Inducible Poly-ADP-Ribose Polymerase (Tiparp) Knockout Mice.

TCDD-inducible poly-ADP-ribose polymerase (TIPARP) is an aryl hydrocarbon receptor (AHR) target gene that functions as part of a negative feedback loop to repress AHR activity. Tiparp-/- mice exhibit increased sensitivity to the toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including lethal wasting syndrome. However, it is not known whether Tiparp-/- mice also exhibit increased sensitivity to other AHR ligands. In this study, we treated male Tiparp-/- or wild type (WT) mice with a single injection of 100 mg/kg 3-methylcholanthrene (3MC). Consistent with TIPARP's role as a repressor of AHR signaling, 3MC-treated Tiparp-/- mice exhibited increased hepatic Cyp1a1 and Cyp1b1 levels compared with WT mice. No 3MC-treated Tiparp-/- mice survived beyond day 16 and the mice exhibited chylous ascites characterized by an accumulation of fluid in the peritoneal cavity. All WT mice survived the 30-day treatment and showed no signs of fluid accumulation. Treated Tiparp-/- mice also exhibited a transient and mild hepatotoxicity with inflammation. 3MC-treated WT, but not Tiparp-/- mice, developed mild hepatic steatosis. Lipid deposits accumulated on the surface of the liver and other abdominal organs in the 3MC-Tiparp-/- mice. Our study reveals that Tiparp-/- mice have increased sensitivity to 3MC-induced liver toxicity, but unlike with TCDD, lethality is due to chylous ascites rather than wasting syndrome.

Liposome mediated-CYP1A1 gene silencing nanomedicine prepared using lipid film-coated proliposomes as a potential treatment strategy of lung cancer.

The occurrence of lung cancer is linked with tobacco smoking, mainly through the generation of polycyclic aromatic hydrocarbons (PAHs). Elevated activity of cytochrome P4501A1 (CYP1A1) plays an important role in the metabolic processing of PAHs and its carcinogenicity. The present work aimed to investigate the role of CYP1A1 gene in PAH-mediated growth and tumor development in vitro and using an in vivo animal model. RNAi strategy was utilized to inhibit the overexpression of CYP1A1 gene using cationic liposomes generated using a lipid film-coated proliposome microparticles. Treatment of PAH-induced human alveolar adenocarcinoma cell line with cationic liposomes carrying CYP1A1 siRNA resulted in down regulation of CYP1A1 mRNA, protein as well as its enzymatic activity, triggering apoptosis and inhibiting multicellular tumor spheroids formation in vitro. Furthermore, silencing of CYP1A1 gene in BALB/c nude xenografts inhibited tumor growth via down regulation of CYP1A1 expression. Altogether, our findings showed that liposome-based gene delivery technology is a viable and stable approach for targeting cancer causing genes such as CY1PA1. This technology facilitated by the use of sugar particles coated with lipid films has demonstrated ability to generate anticancer effects that might be used in the future for therapeutic intervention and treatment of lung cancer.

Mutational landscape in genetically engineered, carcinogen-induced, and radiation-induced mouse sarcoma.

Cancer development is influenced by hereditary mutations, somatic mutations due to random errors in DNA replication, or external factors. It remains unclear how distinct cell-intrinsic and -extrinsic factors impact oncogenesis within the same tissue type. We investigated murine soft tissue sarcomas generated by oncogenic alterations (KrasG12D activation and p53 deletion), carcinogens (3-methylcholanthrene [MCA] or ionizing radiation), and in a novel model combining both factors (MCA plus p53 deletion). Whole-exome sequencing demonstrated distinct mutational signatures in individual sarcoma cohorts. MCA-induced sarcomas exhibited high mutational burden and predominantly G-to-T transversions, while radiation-induced sarcomas exhibited low mutational burden and a distinct genetic signature characterized by C-to-T transitions. The indel to substitution ratio and amount of gene copy number variations were high for radiation-induced sarcomas. MCA-induced tumors generated on a p53-deficient background showed the highest genomic instability. MCA-induced sarcomas harbored mutations in putative cancer-driver genes that regulate MAPK signaling (Kras and Nf1) and the Hippo pathway (Fat1 and Fat4). In contrast, radiation-induced sarcomas and KrasG12Dp53-/- sarcomas did not harbor recurrent oncogenic mutations, rather they exhibited amplifications of specific oncogenes: Kras and Myc in KrasG12Dp53-/- sarcomas, and Met and Yap1 for radiation-induced sarcomas. These results reveal that different initiating events drive oncogenesis through distinct mechanisms.

CD91 on dendritic cells governs immunosurveillance of nascent, emerging tumors.

The immune system detects aberrant, premalignant cells and eliminates them before the development of cancer. Immune cells, including T cells, have been shown to be critical components in eradicating these aberrant cells, and when absent in the host, incidence of cancer increases. Here, we show that CD91, a receptor expressed on antigen-presenting cells, is required for priming immune responses to nascent, emerging tumors. In the absence of CD91, effector immune responses are subdued, and tumor incidence and progression are amplified. We also show that, consequently, tumors that arise in the absence of CD91 express neo-epitopes with indices that are indicative of greater immunogenicity. Polymorphisms in human CD91 that are expected to affect ligand binding are shown to influence antitumor immune responses in cancer patients. This study presents a molecular mechanism for priming immune responses to nascent, emerging tumors that becomes a predictor of cancer susceptibility and progression.

Suppression of Hepatic CYP3A4 Expression and Activity by 3-Methylcholanthrene in Humanized PXR-CAR-CYP3A4/3A7 Mice.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that activate the aryl hydrocarbon receptor, thereby triggering a range of biologic responses, exemplified by the induction of CYP1A1 PAHs can also regulate the expression of members of the CYP3A subfamily, with reports of mainly suppressive effects on mouse hepatic Cyp3a11 expression, but paradoxically both inductive and suppressive effects on human hepatic CYP3A4 expression. Understanding the regulation of CYP3A4 expression by PAHs is important because of the widespread exposure of humans to these chemicals and the central role of the CYP3A4 enzyme in the metabolism of clinically important drugs and endogenous substances. The present study used 3-methylcholanthrene (MC) as a model PAH to characterize the in vivo regulation of CYP3A4 expression and activity in humanized pregnane X receptor-constitutive androstane receptor-CYP3A4/3A7 mice. Adult mice were treated by intraperitoneal injection with MC (80 mg/kg), or corn oil vehicle, and euthanized 24 or 72 hours later. As a positive control response, pronounced induction of hepatic Cyp1a1 by MC was confirmed at both time points in males and females at the mRNA, protein, and catalytic activity levels. Basal hepatic CYP3A4 expression and activity were significantly higher in female versus male mice. MC treatment suppressed hepatic CYP3A4 in female mice at 72 hours postdosing at the mRNA, protein, and catalytic activity levels. A similar response was observed in male mice, although the suppression of CYP3A4 protein levels did not achieve statistical significance. This mouse model will facilitate further studies of the mechanisms and consequences of CYP3A4 suppression by PAHs.

Pulmonary toxicity and lung tumorigenic potential of surrogate metal oxides in gas metal arc welding-stainless steel fume: Iron as a primary mediator versus chromium and nickel.

In 2017, the International Agency for Research on Cancer classified welding fumes as "carcinogenic to humans" (Group 1). Both mild steel (MS) welding, where fumes lack carcinogenic chromium and nickel, and stainless steel (SS) increase lung cancer risk in welders; therefore, further research to better understand the toxicity of the individual metals is needed. The objectives were to (1) compare the pulmonary toxicity of chromium (as Cr(III) oxide [Cr2O3] and Cr (VI) calcium chromate [CaCrO4]), nickel [II] oxide (NiO), iron [III] oxide (Fe2O3), and gas metal arc welding-SS (GMAW-SS) fume; and (2) determine if these metal oxides can promote lung tumors. Lung tumor susceptible A/J mice (male, 4-5 weeks old) were exposed by oropharyngeal aspiration to vehicle, GMAW-SS fume (1.7 mg), or a low or high dose of surrogate metal oxides based on the respective weight percent of each metal in the fume: Cr2O3 + CaCrO4 (366 + 5 µg and 731 + 11 µg), NiO (141 and 281 µg), or Fe2O3 (1 and 2 mg). Bronchoalveolar lavage, histopathology, and lung/liver qPCR were done at 1, 7, 28, and 84 days post-aspiration. In a two-stage lung carcinogenesis model, mice were initiated with 3-methylcholanthrene (10 µg/g; intraperitoneal; 1x) or corn oil then exposed to metal oxides or vehicle (1 x/week for 5 weeks) by oropharyngeal aspiration. Lung tumors were counted at 30 weeks post-initiation. Results indicate the inflammatory potential of the metal oxides was Fe2O3 > Cr2O3 + CaCrO4 > NiO. Overall, the pneumotoxic effects were negligible for NiO, acute but not persistent for Cr2O3 + CaCrO4, and persistent for the Fe2O3 exposures. Fe2O3, but not Cr2O3 + CaCrO4 or NiO significantly promoted lung tumors. These results provide experimental evidence that Fe2O3 is an important mediator of welding fume toxicity and support epidemiological findings and the IARC classification.

Squalene is a potential endocrine modulator in rat: A proof-of-principle study with 3-methylcholanthrene-induced toxicity.

Oestrus urine was proved as a potential endocrine modulator in alleviating the toxicity induced by 3-methylcholanthrene (3-MC) in male rats. We, in this study, aimed to prove the attributing potential of toxicity alleviation to squalene, an oestrus-specific pheromone in rats. A single dose of 3-methylcholanthrene (25 mg/kg BW, i.p.) was administered to male Wistar rats with concurrent exposure to squalene sprayed in bedding material (Group III). Group II rats did receive 3-MC treatment but did not expose to squalene. Group I rats were intact control neither administered 3-MC nor sprayed with squalene. In consequence of 3-MC toxicity, liver and testes weights were increased and the components of blood cells (RBC and WBC count, Hb level) and testosterone concentration were significantly reduced in Group II rats. Moreover, sperm count was reduced and antioxidants (testes and epididymis) were significantly altered. Exposure to squalene in Group III rats comparatively normalised all the variable components towards baseline and reorganised the histological architecture of reproductive tissues that were exacerbated with 3-MC toxicity. This study ultimately proved squalene as a potent molecule in alleviating the toxicity induced by 3-methylcholanthrene.

Role of glutathione S-transferases in detoxification of a polycyclic aromatic hydrocarbon, methylcholanthrene.

Glutathione S-transferases (GSTs), the versatile phase II biotransformation enzymes, metabolize and detoxify a wide variety of toxic chemical compounds like carcinogens, chemotherapeutic drugs, environmental pollutants and oxidative stress products. GSTs are currently of great interest in drug discovery, nanotechnology and biotechnology because of their involvement in many major cellular processes. GSTs, which are either homo or hetero dimeric proteins mediate catalytic binding between glutathione (GSH) and an array of either endogenous or exogenous toxic compounds to form a highly soluble detoxified complex which is then eliminated. Polycyclic aromatic hydrocarbons (PAHs) which are composed of two or more benzene rings bonded as linear, cluster or angular arrangements are used as intermediaries in pharmaceuticals, agricultural products, photographic products, thermosetting plastics, lubricating materials and other chemical products. Foods those cooked at high temperatures by grilling, roasting, frying and smoking are the main sources for the persistent bio-accumulation of PAHs in food chain. The carcinogenic, mutagenic and immunosuppressive effects of PAHs are well established. A well-known polycyclic aromatic hydrocarbon, methylcholanthrene is a potential carcinogenic, neurotoxic, mutagenic and tumour causing agent that is used as an experimental carcinogen in biological research. Methylcholanthrene converts into reactive metabolites when it enters living cells and those reactive metabolites oxidize DNA, RNA, proteins and lipids and form DNA and protein adducts as well. GSTs play major role in the detoxification of reactive metabolites of methylcholanthrene by mediating catalytic binding with GSH to form a highly soluble detoxified complex which is then eliminated. This review summarizes the role of GSTs in the detoxification of a polycyclic aromatic hydrocarbon, methylcholanthrene.

Osteotoxicity of 3-methylcholanthrene in fish.

Many chemicals produced by human activities end up in the aquatic ecosystem causing adverse developmental and reproductive effects in aquatic organisms. There is evidence that some anthropogenic chemicals disturb bone formation and skeletal development but the lack of suitable in vitro and in vivo systems for testing has hindered the identification of underlying mechanisms of osteotoxicity. Several fish systems - an in vitro cell system to study extracellular matrix mineralization and in vivo systems to evaluate bone formation and skeletogenesis - were combined to collect data on the osteotoxic activity of 3-methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon. Anti-mineralogenic effects, increased incidence of skeletal deformities and reduced bone formation and regeneration were observed in zebrafish upon exposure to 3-MC. Pathway reporter array revealed the role of the aryl hydrocarbon receptor 2 (Ahr2) in the mechanisms underlying 3-MC osteotoxicity in mineralogenic cell lines. Analysis of gene expression in zebrafish larvae confirmed the role of Ahr2 in the signaling of 3-MC toxicity. It also indicated a possible complementary action of the pregnane X receptor (Pxr) in the regulation of genes involved in bone cell activity and differentiation but also in xenobiotic metabolism. Data reported here demonstrated the osteotoxicity of 3-MC but also confirmed the suitability of fish systems to gain insights into the toxic mechanisms of compounds affecting skeletal and bone formation.

The transformics assay: first steps for the development of an integrated approach to investigate the malignant cell transformation in vitro.

The development of alternative methods to animal testing is a priority in the context of regulatory toxicology. Carcinogenesis is a field where the demand for alternative methods is particularly high. The standard rodent carcinogenicity bioassay requires a large use of animals, high costs, prolonged duration and shows several limitations, which can affect the comprehension of the human relevance of animal carcinogenesis. The cell transformation assay (CTA) has long been debated as a possible in vitro test to study carcinogenesis. This assay provides an easily detectable endpoint of oncotransformation, which can be used to anchor the exposure to the acquisition of the malignant phenotype. However, the current protocols do not provide information on either molecular key events supporting the carcinogenesis process, nor the mechanism of action of the test chemicals. In order to improve the use of this assay in the integrated testing strategy for carcinogenesis, we developed the transformics method, which combines the CTA and transcriptomics, to highlight the molecular steps leading to in vitro malignant transformation. We studied 3-methylcholanthrene (3-MCA), a genotoxic chemical able to induce in vitro cell transformation, at both transforming and subtransforming concentrations in BALB/c 3T3 cells and evaluated the gene modulation at critical steps of the experimental protocol. The results gave evidence for the potential key role of the immune system and the possible involvement of the aryl hydrocarbon receptor (AhR) pathway as the initial steps of the in vitro transformation process induced by 3-MCA, suggesting that the initiating events are related to non-genotoxic mechanisms.