In Vivo Anti-Hepatocellular Carcinoma Effects of the Chloroform Root Extract of Clausena excavata Burm.

Liver cancer is the most common cancer among males in Africa. The disease has a poor prognosis and its treatment is associated with toxicity and resistance. For this reason, numerous herbal combinations are being subjected to anticancer screening to circumvent the shortcomings of the conventional anticancer drugs. In the current study, the in vivo anti-cancer effects of the chloroform root extract of the herb, Clausena excavata Burm were investigated. Liver cancer was induced in mice by a single intraperitoneal injection of diethylnitrosamine (DEN) followed by oral administration of the promoter of carcinogenesis, 2-aminoacetyl fluorine that was mixed with the mice feed. The cytotoxicity of the root extract of C. excavata on liver cancer cells was investigated using liver enzyme, histology, DNA fragmentation and caspases assays. Real time qPCR was conducted to evaluate the effect of the extract on apoptotic genes. The findings revealed that the extract of C. excavata significantly decreased the progression of hepatocarcinogenesis and the toxicity-induced production of the liver enzymes, alanine and aspartate aminotransferases. The histological analyses of the liver tissues revealed evidence of apoptotic cell death. The extract also provoked significant (p < .05) expressions of caspase 9 protein and gene as well as other apoptotic genes (P53, P27, Apaf-1, cytochrome C, bax and bid). Therefore, we postulate that the chloroform root extract of C. excavata induces apoptosis of liver cancer in mice.

Pantoprazole suppresses carcinogenesis and growth of hepatocellular carcinoma by inhibiting glycolysis and Na+/H+ exchange.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. The prevention and therapy for this deadly disease remain a global medical challenge. In this study, we investigated the effect of pantoprazole (PPZ) on the carcinogenesis and growth of HCC. Both diethylnitrosamine (DEN) plus CCl4-induced and DEN plus high fat diet (HFD)-induced HCC models in mice were established. Cytokines and cell proliferation-associated gene in the liver tissues of mice and HCC cells were analyzed. Cellular glycolysis and Na+/H+ exchange activity were measured. The preventive administration of pantoprazole (PPZ) at a clinically relevant low dose markedly suppressed HCC carcinogenesis in both DEN plus CCl4-induced and HFD-induced murine HCC models, whereas the therapeutic administration of PPZ at the dose suppressed the growth of HCC. In the liver tissues of PPZ-treated mice, inflammatory cytokines, IL1, CXCL1, CXCL5, CXCL9, CXCL10, CCL2, CCL5, CCL6, CCL7, CCL20, and CCL22, were reduced. The administration of CXCL1, CXCL5, CCL2, or CCL20 all reversed PPZ-suppressed DEN plus CCL4-induced HCC carcinogenesis in mice. PPZ inhibited the expressions of CCNA2, CCNB2, CCNE2, CDC25C, CDCA5, CDK1, CDK2, TOP2A, TTK, AURKA, and BIRC5 in HCC cells. Further results showed that PPZ reduced the production of these inflammatory cytokines and the expression of these cell proliferation-associated genes through the inhibition of glycolysis and Na+/H+ exchange. In conclusion, PPZ suppresses the carcinogenesis and growth of HCC, which is related to inhibiting the production of inflammatory cytokines and the expression of cell proliferation-associated genes in the liver through the inhibition of glycolysis and Na+/H+ exchange.

Genotoxicity assessments of N-nitrosoethylisopropylamine (NEIPA) and N-nitrosodiisopropylamine (NDIPA) in the C57BL/6J mouse.

N-Nitrosamines, known as drug impurities and suspected carcinogens, have drawn significant public concern. In response to drug regulatory needs, the European Medicines Agency (EMA) has previously proposed a carcinogenic potency categorization approach based on the N-nitrosamine α-hydroxylation hypothesis, i.e., that N-nitrosamine mutagenicity increases with the number of α-hydrogen atoms. However, this structure-activity relationship has not been fully tested in vivo. NEIPA (N-nitrosoethylisopropylamine) and NDIPA (N-nitrosodiisopropylamine) are small N-Nitrosamines with similar structures, differing in that the former compound has an additional α-hydrogen atom. In this study, NEIPA and NEIPA doses, 25-100 mg/kg, were administered orally to C57BL/6 J mice for seven consecutive days, and their mutation and DNA damage effects were compared. Compared with NDIPA, the mutagenicity and DNA damage potencies of NEIPA (which contains one more α-hydrogen) were much greater. These differences may be related to their distinct metabolic pathways and target organs. This case study confirms the role of α-hydroxyl modification in the mutagenicity of nitrosamines, with oxidation at the α-hydrogen being a crucial step in the formation of mutagens from N-Nitrosamines, and can inform mutagenicity risk assessment and the formulation of regulatory standards for N-nitrosamine impurities.

SMARCA4 (BRG1) activates ABCC3 transcription to promote hepatocellular carcinogenesis.

AIMS: Hepatocellular carcinoma (HCC) is a lead cause of cancer-related deaths. In the present study we investigated the role of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, in HCC the pathogenesis focusing on identifying novel transcription targets. METHODS AND MATERIALS: Hepatocellular carcinogenesis was modeled in mice by diethylnitrosamine (DEN). Cellular transcriptome was evaluated by RNA-seq. RESULTS: Hepatocellular carcinoma was appreciably retarded in BRG1 knockout mice compared to wild type littermates. Transcriptomic analysis identified ATP Binding Cassette Subfamily C Member 3 (ABCC3) as a novel target of BRG1. BRG1 over-expression in BRG1low HCC cells (HEP1) up-regulated whereas BRG1 depletion in BRG1high HCC cells (SNU387) down-regulated ABCC3 expression. Importantly, BRG1 was detected to directly bind to the ABCC3 promoter to activate ABCC3 transcription. BRG1 over-expression in HEP1 cells promoted proliferation and migration, both of which were abrogated by ABCC3 silencing. On the contrary, BRG1 depletion in SNU387 cells decelerated proliferation and migration, both of which were rescued by ABCC3 over-expression. Importantly, high BRG1/ABCC3 expression predicted poor prognosis in HCC patients. Mechanistically, ABCC3 regulated hepatocellular carcinogenesis possibly by influencing lysosomal homeostasis. SIGNIFICANCE: In conclusion, our data suggest that targeting BRG1 and its downstream target ABCC3 can be considered as a reasonable approach for the intervention of hepatocellular carcinoma.

Proteomic analysis of DEN and CCl4-induced hepatocellular carcinoma mouse model.

Hepatocellular carcinoma (HCC) seriously threatens human health, mostly developed from liver fibrosis or cirrhosis. Since diethylnitrosamine (DEN) and carbon tetrachloride (CCl4)-induced HCC mouse model almost recapitulates the characteristic of HCC with fibrosis and inflammation, it is taken as an essential tool to investigate the pathogenesis of HCC. However, a comprehensive understanding of the protein expression profile of this model is little. In this study, we performed proteomic analysis of this model to elucidate its proteomic characteristics. Compared with normal liver tissues, 432 differentially expressed proteins (DEPs) were identified in tumor tissues, among which 365 were up-regulated and 67 were down-regulated. Through Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), protein-protein interaction networks (PPI) analysis and Gene-set enrichment analysis (GSEA) analysis of DEPs, we identified two distinguishing features of DEN and CCl4-induced HCC mouse model in protein expression, the upregulation of actin cytoskeleton and branched-chain amino acids metabolic reprogramming. In addition, matching DEPs from the mouse model to homologous proteins in the human HCC cohort revealed that the DEN and CCl4-induced HCC mouse model was relatively similar to the subtype of HCC with poor prognosis. Finally, combining clinical information from the HCC cohort, we screened seven proteins with prognostic significance, SMAD2, PTPN1, PCNA, MTHFD1L, MBOAT7, FABP5, and AGRN. Overall, we provided proteomic data of the DEN and CCl4-induced HCC mouse model and highlighted the important proteins and pathways in it, contributing to the rational application of this model in HCC research.

Targeting the PI3K/pAKT/mTOR/NF-κB/FOXO3a signaling pathway for suppressing the development of hepatocellular carcinoma in rats: Role of the natural remedic Suaeda vermiculata forssk.

Liver malignancy is well recognized as a prominent health concern, with numerous treatment options available. Natural products are considered a renewable source, providing inspiring chemical moieties that could be used for cancer treatment. Suaeda vermiculata Forssk has traditionally been employed for management of hepatic conditions, including liver inflammation, and liver cirrhosis, as well as to improve general liver function. The findings of our earlier study demonstrated encouraging in vivo hepatoprotective benefits against liver injury generated by paracetamol and carbon tetrachloride. Additionally, Suaeda vermiculata Forssk exhibited cytotoxic activities in vitro against Hep-G2 cell lines and cell lines resistant to doxorubicin. The present investigation aimed to examine the potential in vivo hepatoprotective efficacy of Suaeda vermiculata Forssk extract (SVE) against hepatocellular carcinoma induced by diethylnitrosamine (DENA) in rats. The potential involvement of the PI3K/AKT/mTOR/NF-κB pathway was addressed. Sixty adult male albino rats were allocated into five groups randomly (n = 10). First group received a buffer, whereas second group received SVE only, third group received DENA only, and fourth and fifth groups received high and low doses of SVE, respectively, in the presence of DENA. Liver toxicity and tumor markers (HGFR, p-AKT, PI3K, mTOR, NF-κB, FOXO3a), apoptosis markers, and histopathological changes were analyzed. The current results demonstrated that SVE inhibited PI3K/AKT/mTOR/NF-κB pathway as well as increased expression of apoptotic parameters and FOXO3a levels, which were deteriorated by DENA treatment. Furthermore, SVE improved liver toxicity markers and histopathological changes induced by DENA administration. This study provided evidence for the conventional hepatoprotective properties attributed to SV and investigated the underlying mechanism by which its extract, SVE, could potentially serve as a novel option for hepatocellular carcinoma (HCC) treatment derived from a natural source.

An adenosine derivative promotes mitochondrial supercomplexes reorganization and restoration of mitochondria structure and bioenergetics in a diethylnitrosamine-induced hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) progression is associated with dysfunctional mitochondria and bioenergetics impairment. However, no data about the relationship between mitochondrial supercomplexes (hmwSC) formation and ATP production rates in HCC are available. Our group has developed an adenosine derivative, IFC-305, which improves mitochondrial function, and it has been proposed as a therapeutic candidate for HCC. We aimed to determine the role of IFC-305 on both mitochondrial structure and bioenergetics in a sequential cirrhosis-HCC model in rats. Our results showed that IFC-305 administration decreased the number and size of liver tumors, reduced the expression of tumoral markers, and reestablished the typical architecture of the hepatic parenchyma. The livers of treated rats showed a reduction of mitochondria number, recovery of the mtDNA/nDNA ratio, and mitochondrial length. Also, IFC-305 increased cardiolipin and phosphatidylcholine levels and promoted hmwSC reorganization with changes in the expression levels of hmwSC assembly-related genes. IFC-305 in HCC modified the expression of several genes encoding elements of electron transport chain complexes and increased the ATP levels by recovering the complex I, III, and V activity. We propose that IFC-305 restores the mitochondrial bioenergetics in HCC by normalizing the quantity, morphology, and function of mitochondria, possibly as part of its hepatic restorative effect.

Compromised very-low density lipoprotein induced polyunsaturated triglyceride accumulation in N-nitrosodiethylamine-induced hepatic steatosis.

N-Nitrosodiethylamine (NDEA), a carcinogen in some foods and medications, is linked to liver damage similar to non-alcoholic fatty liver disease (NAFLD). This study explores how NDEA disrupts liver lipid metabolism. Sprague-Dawley rats were given two doses of NDEA (100 mg/kg) orally, 24 h apart. Liver response was assessed through tissue staining, blood tests, and biochemical markers, including fatty acids, lipid peroxidation, and serum very-low density lipoprotein (VLDL) levels. Additionally, lipidomic analysis of liver tissues and serum was performed. The results indicated significant hepatic steatosis (fat accumulation in the liver) following NDEA exposure. Blood analysis showed signs of inflammation and liver damage. Biochemical tests revealed decreased liver protein synthesis and specific enzyme alterations, suggesting liver cell injury but maintaining mitochondrial function. Increased fatty acid levels without a rise in lipid peroxidation were observed, indicating fat accumulation. Lipidomic analysis showed increased polyunsaturated triglycerides in the liver and decreased serum VLDL, implicating impaired VLDL transport in liver dysfunction. In conclusion, NDEA exposure disrupts liver lipid metabolism, primarily through the accumulation of polyunsaturated triglycerides and impaired fat transport. These findings provide insight into the mechanisms of NDEA-induced liver injury and its progression to hepatic steatosis.

Diethylnitrosamine Induction of Hepatocarcinogenesis in Mice.

Diethylnitrosamine (DEN) is a chemical hepatocarcinogenic agent that triggers a large array of oncogenic mutations after a single injection. Initiated hepatocytes subsequently undergo clonal expansion within a proliferative environment, rendering the DEN model a comprehensive carcinogen. In rodent studies, DEN finds extensive utility in experimental liver cancer research, mimicking several aspects of human hepatocellular carcinoma (HCC), including angiogenesis, metabolic reprogramming, immune exhaustion, and the ability to metastasize. Beyond the wealth of scientific insights gleaned from this model, the objective of this chapter is to review morphological, genomic, and immunological characteristics associated to DEN-induced HCC. Furthermore, this chapter provides a detailed procedural guide to effectively induce hepatocarcinogenesis in mice through a single intraperitoneal injection of DEN.

Diethylnitrosamine-Induced Liver Tumorigenesis in Mice Under High-Hat High-Sucrose Diet: Stepwise High-Resolution Ultrasound Imaging and Histopathological Correlations.

The hepatotoxic N-nitroso compound diethylnitrosamine (DEN) administered intraperitoneally (i.p.) induces liver neoplasms in rodents that reproducibly recapitulate some aspects of human hepatocarcinogenesis. In particular, DEN drives the stepwise formation of pre-neoplastic and neoplastic (benign or malignant) hepatocellular lesions reminiscent of the initiation-promotion-progression sequence typical of chemical carcinogenesis. In humans, the development of hepatocellular carcinoma (HCC) is also a multi-step process triggered by continuous hepatocellular injury, chronic inflammation, and compensatory hyperplasia that fuel the emergence of dysplastic liver lesions followed by the formation of early HCC. The DEN-induced liver tumorigenesis model represents a versatile preclinical tool that enables the study of many tumor development modifiers (genetic background, gene knockout or overexpression, diets, pollutants, or drugs) with a thorough follow-up of the multistage process on live animals by means of high-resolution imaging. Here, we provide a comprehensive protocol for the induction of hepatocellular neoplasms in wild-type C57BL/6J male mice following i.p. DEN injection (25 mg/kg) at 14 days of age and 36 weeks feeding of a high-fat high-sucrose (HFHS) diet. We emphasize the use of ultrasound liver imaging to follow tumor development and provide histopathological correlations. We also discuss the extrinsic and intrinsic factors known to modify the course of liver tumorigenesis in this model.

Loss of hepatic Sirt7 accelerates diethylnitrosamine (DEN)-induced formation of hepatocellular carcinoma by impairing DNA damage repair.

The mammalian sirtuin family (SIRT1-SIRT7) has shown diverse biological roles in the regulation and maintenance of genome stability under genotoxic stress. SIRT7, one of the least studied sirtuin, has been demonstrated to be a key factor for DNA damage response (DDR). However, conflicting results have proposed that Sirt7 is an oncogenic factor to promote transformation in cancer cells. To address this inconsistency, we investigated properties of SIRT7 in hepatocellular carcinoma (HCC) regulation under DNA damage and found that loss of hepatic Sirt7 accelerated HCC progression. Specifically, the number, size, and volume of hepatic tumor colonies in diethylnitrosamine (DEN) injected Sirt7-deficient liver were markedly enhanced. Further, levels of HCC progression markers and pro-inflammatory cytokines were significantly elevated in the absence of hepatic Sirt7, unlike those in the control. In chromatin, SIRT7 was stabilized and colocalized to damage site by inhibiting the induction of γH2AX under DNA damage. Together, our findings suggest that SIRT7 is a crucial factor for DNA damage repair and that hepatic loss-of-Sirt7 can promote genomic instability and accelerate HCC development, unlike early studies describing that Sirt7 is an oncogenic factor [BMB Reports 2024; 57(2): 98-103].

Linagliptin, a DPP-4 inhibitor, activates AMPK/FOXO3a and suppresses NFκB to mitigate the debilitating effects of diethylnitrosamine exposure in rat liver: Novel mechanistic insights.

Accumulating evidence suggests that dysregulation of FOXO3a plays a significant role in the progression of various malignancies, including hepatocellular carcinoma (HCC). FOXO3a inactivation, driven by oncogenic stimuli, can lead to abnormal cell growth, suppression of apoptosis, and resistance to anticancer drugs. Therefore, FOXO3a emerges as a potential molecular target for the development of innovative treatments in the era of oncology. Linagliptin (LNGTN), a DPP-4 inhibitor known for its safe profile, has exhibited noteworthy anti-inflammatory and anti-oxidative properties in previous in vivo studies. Several potential molecular mechanisms have been proposed to explain these effects. However, the capacity of LNGTN to activate FOXO3a through AMPK activation has not been investigated. In our investigation, we examined the potential repurposing of LNGTN as a hepatoprotective agent against diethylnitrosamine (DENA) intoxication. Additionally, we assessed LNGTN's impact on apoptosis and autophagy. Following a 10-week administration of DENA, the liver underwent damage marked by inflammation and early neoplastic alterations. Our study presents the first experimental evidence demonstrating that LNGTN can reinstate the aberrantly regulated FOXO3a activity by elevating the nuclear fraction of FOXO3a in comparison to the cytosolic fraction, subsequent to AMPK activation. Moreover, noteworthy inactivation of NFκB induced by LNGTN was observed. These effects culminated in the initiation of apoptosis, the activation of autophagy, and the manifestation of anti-inflammatory, antiproliferative, and antiangiogenic outcomes. These effects were concomitant with improved liver function and microstructure. In conclusion, our findings open new avenues for the development of novel therapeutic strategies targeting the AMPK/FOXO3a signaling pathway in the management of chronic liver damage.

Effectiveness of cannabidiol (CBD) on histopathological changes and gene expression in hepatocellular carcinoma (HCC) model in male rats: the role of Hedgehog (Hh) signaling pathway.

The third most prevalent malignancy to cause mortality is hepatocellular carcinoma (HCC). The Hedgehog (Hh) signaling pathway is activated by binding to the transmembrane receptor Patched-1 (PTCH-1), which depresses the transmembrane G protein-coupled receptor Smoothened (SMO). This study was performed to examine the preventative and therapeutic effects of cannabidiol in adult rats exposed to diethyl nitrosamine (DENA)-induced HCC.A total of 50 male rats were divided into five groups of 10 rats each. Group I was the control group. Group II received intraperitoneal (IP) injections of DENA for 14 weeks. Group III included rats that received cannabidiol (CBD) orally (3-30 mg/kg) for 2 weeks and DENA injections for 14 weeks. Group IV rats received oral CBD for 2 weeks before 14 weeks of DENA injections. Group V included rats that received CBD orally for 2 weeks after their last injection of DENA. Measurements were made for alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and alpha fetoprotein (AFP). Following total RNA extraction, Smo, Hhip, Ptch-1, and Gli-1 expressions were measured using quantitative real-time polymerase chain reaction (qRT-PCR). A histopathological analysis of liver tissues was performed.The liver enzymes, oxidant-antioxidant state, morphological, and molecular parameters of the adult male rat model of DENA-induced HCC showed a beneficial improvement after CBD administration. In conclusion, by focusing on the Hh signaling system, administration of CBD showed a beneficial improvement in the liver enzymes, oxidant-antioxidant status, morphological, and molecular parameters in the DENA-induced HCC in adult male rats.

Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

Structural abnormality of hepatic glycogen in rat liver with diethylnitrosamine-induced carcinogenic injury.

Growing evidence confirms associations between glycogen metabolic re-wiring and the development of liver cancer. Previous studies showed that glycogen structure changes abnormally in liver diseases such as cystic fibrosis, diabetes, etc. However, few studies focus on glycogen molecular structural characteristics during liver cancer development, which is worthy of further exploration. In this study, a rat model with carcinogenic liver injury induced by diethylnitrosamine (DEN) was successfully constructed, and hepatic glycogen structure was characterized. Compared with glycogen structure in the healthy rat liver, glycogen chain length distribution (CLD) shifts towards a short region. In contrast, glycogen particles were mainly present in small-sized ß particles in DEN-damaged carcinogenic rat liver. Comparative transcriptomic analysis revealed significant expression changes of genes and pathways involved in carcinogenic liver injury. A combination of transcriptomic analysis, RT-qPCR, and western blot showed that the two genes, Gsy1 encoding glycogen synthase and Gbe1 encoding glycogen branching enzyme, were significantly altered and might be responsible for the structural abnormality of hepatic glycogen in carcinogenic liver injury. Taken together, this study confirmed that carcinogenic liver injury led to structural abnormality of hepatic glycogen, which provided clues to the future development of novel drug targets for potential therapeutics of carcinogenic liver injury.

Obesity phenotype induced by high-fat diet promotes diethylnitrosamine (DEN)-induced precancerous lesions by M1 macrophage polarization in mice liver.

Liver precancerous lesions are the key to improving the efficacy of cancer treatment because of the extremely poor prognosis of HCC patients in moderate and late stages. Obesity-related HCC progression is closely related to the inflammatory microenvironment, in which macrophages are one of the major constituents. In the present study, we ask whether obesity promotes diethylnitrosamine (DEN)-induced precancerous lesions by M1 macrophage polarization. First, an association between obesity and liver precancerous lesions was determined by histopathological observations, immunochemistry and immunoblotting. The characteristics of early precancerous lesions (trabecular thickening) appeared earlier eight weeks in obese mice than in normal diet mice after DEN induction. The glutathione S-transferase placental-1 (Gstp 1) and alpha-fetoprotein (AFP) expression in obese mice after DEN induction was higher than that in the same period after DEN injection in normal diet mice. Furthermore, there was a significant increase in the total macrophage number (F4/80+) of DEN and M1 macrophage number (CD86+F4/80+) in obese mice compared with that in normal diet mice. Besides, the expressions of four pro-inflammatory factors in DEN-induced obese mice were significantly higher compared with that in normal diet mice. Additionally, angiogenesis was revealed by immunostaining assay to be associated with the inflammatory response. All the results demonstrate that obesity promotes DEN-induced precancerous lesions by inducing M1 macrophage polarization and angiogenesis.

Improvement of rat hepatocellular carcinoma model induced by diethylnitrosamine.

OBJECTIVE: To construct a new diethylnitrosamine (DEN)-induced rat hepatocellular carcinoma (HCC) model with short induction time, high incidence, and survival rate. METHODS: 60 male Sprague-Dawley rats were randomly divided into 4 groups: the control group, the model A (MA) group, the model B (MB) group, and the model C (MC) group. The control group was intraperitoneally injected with 0.9% saline for 6 weeks. The MA group was injected with the DEN solution at 30 mg/kg three times a week for 6 weeks. The MB group was injected with the DEN solution at 30 mg/kg three times a week for 6 weeks, and discontinued the induction for 2 weeks. The MC group was injected with the DEN solution at 30 mg/kg three times a week for 8 weeks. The levels of albumin (ALB), alanine transaminase (ALT), and aspartate aminotransferase (AST) in serum were assayed. Meanwhile, the pathological conditions, apoptosis of hepatocytes, expression of NF-κBp65, and the reactive oxygen species level were detected. RESULTS: All rats in the control group and the MA group survived, and none of the rats occurred HCC. HCC occurred in rats of the MB group and the MC group. The serum ALB level in the MB group was higher than that in the MC group. The serum ALT and AST levels and the number of proliferating and apoptotic hepatocyte cells in the MB group were lower than those in the MC group. The expression of ROS- and NF-κBp6- positive cells in the MA group, MB group, and MC group were significantly higher than that of the control group. CONCLUSION: This study developed a new DEN-induced rat HCC model with short induction time, high incidence, and survival rate. NF-κB pathway may be one of the main pathways involved in the development of this model.

Feasibility of NDEA formation control from DEDTC in chlorination/chloramination by pre-ozonation: Mechanisms and influencing factors.

N-nitrosodiethylamine (NDEA), which is the most toxic nitrosamine among the 9 detected species, has been widely detected in drinking water. Amines containing diethylamine (DEA) groups in the structure would generate NDEA during the disinfection processes. The aim of this study was to evaluate the feasibility of reducing NDEA formation from a commonly used dithiocarbamate pesticide sodium diethyldithiocarbamate (DEDTC) in subsequent chlorination and chloramination by pre-ozonation. The results demonstrated that NDEA could be generated directly during ozonation, its amounts increased from 0 to 14.34 µg/L with increasing ozone dosages (0-4 mg/L), which was higher than that chlorination (2.68 µg/L) and chloramination (4.91 µg/L) when the initial concentration of DEDTC was 20 µM. Pre-ozonation significantly raised NDEA formation from 2.68 to15.32 µg/L in subsequent chlorination; and that from 4.91 to 9.54 µg/L during subsequent chloramination processes. The addition of •OH scavenger tert-butanol (tBA) increased the production of NDEA from 8.14 to 20.80 µg/L during ozonation, and that from 6.76 to17.98 µg/L in O3/HClO process, 8.74 to 17.33 µg/L in O3/NH2Cl process. Except for NO3- and CO32-, most of the co-existing substances promoted NDEA generation from DEDTC under disinfection conditions. Based on the results of Gaussian theory calculations, GC/MS and UPLC-Q-TOFMS analysis, the influencing mechanisms of pre-ozonation on NDEA generation in the subsequent disinfection process were proposed. In addition, not only acute/chronic toxicity calculation but also luminescent bacteria test was performed to assess the possibility of pre-ozonation on the risk control of DEDTC. The research results fill a gap in the control of NDEA pollution and help to develop a safer ozone oxidation technology.

Modulatory mechanisms of copperII-albumin complex toward N-nitrosodiethylamine-induced neurotoxicity in mice via regulating oxidative damage, inflammatory, and apoptotic signaling pathways.

N-nitrosodiethylamine (ND) is an extremely toxic unavoidable environmental contaminant. CopperII-albumin (CuAB) complex, a newly developed Cu complex, showed antioxidant and anti-inflammatory potential. Hereby, we explored the plausible neuroprotective role of CuAB complex toward ND-evoked neurotoxicity in mice. Twenty-four male mice were sorted into 4 groups (6 mice each). Control group, mice were administered oral distilled water; and CuAB group, mice received CuAB complex at a dose of 817 µg/kg orally, three times weekly. In ND group, ND was given intraperitoneally (50 mg/kg body weight, once weekly for 6 w). CuAB+ND group, mice were administered a combination of CuAB and ND. The brain was quickly extracted upon completion of the experimental protocol for the evaluation of the oxidative/antioxidative markers, inflammatory cytokines, and histopathological examination. Oxidative stress was induced after ND exposure indicated by a reduction in GSH and SOD1 level, with increased MDA level. In addition, decreased expression of SOD1 proteins, Nrf2, and 5-HT mRNA expression levels were noticed. An apoptotic cascade has also been elicited, evidenced by overexpression of Cyt c, Cl. Casp 3. In addition, increased regulation of proinflammatory genes (TNF-α, IL-6, iNOS, Casp1, and NF-κB (p65/p50); besides, increment of protein expression of P-IKBα and reduced expression of IKBα. Pretreatment with CuAB complex significantly ameliorated ND neuronal damage. Our results recommend CuAB complex supplementation because it exerts neuroprotective effects against ND-induced toxicity.

Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.