Mode of action of dieldrin-induced liver tumors: application to human risk assessment.

Dieldrin is an organochlorine insecticide that was widely used until 1970 when its use was banned because of its liver carcinogenicity in mice. Several long-term rodent bioassays have reported dieldrin to induce liver tumors in in several strains of mice, but not in rats. This article reviews the available information on dieldrin liver effects and performs an analysis of mode of action (MOA) and human relevance of these liver findings. Scientific evidence strongly supports a MOA based on CAR activation, leading to alterations in gene expression, which result in increased hepatocellular proliferation, clonal expansion leading to altered hepatic foci, and ultimately the formation of hepatocellular adenomas and carcinomas. Associative events include increased liver weight, centrilobular hypertrophy, increased expression of Cyp2b10 and its resulting increased enzymatic activity. Other associative events include alterations of intercellular gap junction communication and oxidative stress. Alternative MOAs are evaluated and shown not to be related to dieldrin administration. Weight of evidence shows that dieldrin is not DNA reactive, it is not mutagenic, and it is not genotoxic in general. Furthermore, activation of other pertinent nuclear receptors, including PXR, PPARα, AhR, and estrogen are not related to dieldrin-induced liver tumors nor is there liver cytotoxicity. In previous studies, rats, dogs, and non-human primates did not show increased cell proliferation or production of pre-neoplastic or neoplastic lesions following dieldrin treatment. Thus, the evidence strongly indicates that dieldrin-induced mouse liver tumors are due to CAR activation and are specific to the mouse, which are qualitatively not relevant to human hepatocarcinogenesis. Thus, there is no carcinogenic risk to humans. This conclusion is also supported by a lack of positive epidemiologic findings for evidence of liver carcinogenicity. Based on current understanding of the mode of action of dieldrin-induced liver tumors in mice, the appropriate conclusion is that dieldrin is a mouse specific liver carcinogen and it does not pose a cancer risk to humans.

Nicotine or marijuana vaping exposure during pregnancy and altered immune responses in offspring.

Electronic nicotine delivery systems (ENDS) - which include electronic cigarettes or e-cigarettes, or simply e-cigs, and marijuana vaping have become increasingly popular. ENDS devices have been established as one of the tobacco quit methods and promoted to be safer compared to traditional tobacco cigarettes. Emerging evidence demonstrates that e-cigarette and marijuana vape use can be harmful, with potential associations with cancer. Herein, we summarize the level of evidence to date for altered immune response, with a focus on cancer risks in the offspring after maternal use of, or aerosol exposures from, ENDS or marijuana vape during pregnancy. From 27 published articles retrieved from PubMed, we sought to find out identified carcinogens in ENDS aerosols and marijuana vapor, which cross the placental barrier and can increase cancer risk in the offspring. Carcinogens in vaping aerosols include aldehydes, metals, tobacco-specific nitrosamines, tobacco alkaloids, polycyclic aromatic hydrocarbons, and volatile organic compounds. Additionally, there was only one passive vaping exposure case study on a human fetus, which noted that glycerol, aluminum, chromium, nickel, copper, zinc, selenium, and lead crossed from the mother to the offspring's cord blood. The carcinogens (metals) in that study were at lower concentrations compared to the mother's biological matrices. Lastly, we observed that in utero exposures to ENDS-associated chemicals can occur in vital organs such as the lungs, kidneys, brain, bladder, and heart. Any resulting DNA damage increases the risk of tumorigenesis. Future epidemiological studies are needed to examine the effects of passive aerosol exposures from existing and emerging electronic nicotine and marijuana products on developing offspring to cancer.

Evaluating the mode of action of perfluorooctanoic acid-induced liver tumors in male Sprague-Dawley rats using a toxicogenomic approach.

The mode of action (MOA) underlying perfluorooctanoic acid (PFOA)-induced liver tumors in rats is proposed to involve peroxisome proliferator-activated receptor α (PPARα) agonism. Despite clear PPARα activation evidence in rodent livers, the mechanisms driving cell growth remain elusive. Herein, we used dose-responsive apical endpoints and transcriptomic data to examine the proposed MOA. Male Sprague-Dawley rats were treated with 0, 1, 5, and 15 mg/kg PFOA for 7, 14, and 28 days via oral gavage. We showed PFOA induced hepatomegaly along with hepatocellular hypertrophy in rats. PPARα was activated in a dose-dependent manner. Toxicogenomic analysis revealed six early biomarkers (Cyp4a1, Nr1d1, Acot1, Acot2, Ehhadh, and Vnn1) in response to PPARα activation. A transient rise in hepatocellular DNA synthesis was demonstrated while Ki-67 labeling index showed no change. Transcriptomic analysis indicated no significant enrichment in pathways related to DNA synthesis, apoptosis, or the cell cycle. Key cyclins including Ccnd1, Ccnb1, Ccna2, and Ccne2 were dose-dependently suppressed by PFOA. Oxidative stress and the nuclear factor-κB signaling pathway were unaffected. Overall, evidence for PFOA-induced hepatocellular proliferation was transient within the studied timeframe. Our findings underscore the importance of considering inter-species differences and chemical-specific effects when evaluating the carcinogenic risk of PFOA in humans.

Assessment of the mode of action of perchloroethylene-induced mouse liver tumors.

Perchloroethylene (PCE) is used as a solvent and chemical intermediate. Following chronic inhalation exposure, PCE selectively induced liver tumors in mice. Understanding the mode of action (MOA) for PCE carcinogenesis in mice is important in defining its possible human cancer risk. The proposed MOA is based on the extensive examination of the peer-reviewed studies that have assessed the mouse liver effects of PCE and its major oxidative metabolite trichloroacetic acid (TCA). Similar to PCE, TCA has also been demonstrated to liver tumors selectively in mice following chronic exposure. The Key Events (KE) of the proposed PCE MOA involve oxidative metabolism of PCE to TCA [KE 1]; activation of the peroxisome proliferator-activated receptor alpha (PPARα) [KE 2]; alteration in hepatic gene expression including cell growth pathways [KE 3]; increase in cell proliferation [KE 4]; selective clonal expansion of hepatic preneoplastic foci [KE 5]; and formation of hepatic neoplasms [KE 6]. The scientific evidence supporting the PPARα MOA for PCE is strong and satisfies the requirements for a MOA analysis. The PPARα liver tumor MOA in rodents has been demonstrated not to occur in humans; thus, human liver cancer risk to PCE is not likely.

Kinetically-derived maximal dose (KMD) indicates lack of human carcinogenicity of ethylbenzene.

The kinetically-derived maximal dose (KMD) is defined as the maximal external dose at which kinetics are unchanged relative to lower doses, e.g., doses at which kinetic processes are not saturated. Toxicity produced at doses above the KMD can be qualitatively different from toxicity produced at lower doses. Here, we test the hypothesis that neoplastic lesions reported in the National Toxicology Program's (NTP) rodent cancer bioassay with ethylbenzene are a high-dose phenomenon secondary to saturation of elimination kinetics. To test this, we applied Bayesian modeling on kinetic data for ethylbenzene from rats and humans to estimate the Vmax and Km for the Michaelis-Menten equation that governs the elimination kinetics. Analysis of the Michaelis-Menten elimination curve generated from those Vmax and Km values indicated KMD ranges for venous ethylbenzene of 8-17 mg/L in rats and 10-18 mg/L in humans. Those venous concentrations are produced by inhalation concentrations of around 200 ppm ethylbenzene, which is well above typical human exposures. These KMD estimates support the hypothesis that neoplastic lesions seen in the NTP rodent bioassay occur secondary to saturation of ethylbenzene elimination pathways and are not relevant for human risk assessment. Thus, ethylbenzene does not pose a credible cancer risk to humans under foreseeable exposure conditions. Cancer risk assessments focused on protecting human health should avoid endpoint data from rodents exposed to ethylbenzene above the KMD range and future toxicological testing should focus on doses below the KMD range.

Quantitative Integration of Mode of Action Information in Dose-Response Modeling and POD Estimation for Nonmutagenic Carcinogens: A Case Study of TCDD.

BACKGROUND: Traditional dose-response assessment applies different low-dose extrapolation methods for cancer and noncancer effects and assumes that all carcinogens are mutagenic unless strong evidence suggests otherwise. Additionally, primarily focusing on one critical effect, dose-response modeling utilizes limited mode of action (MOA) data to inform low-dose risk. OBJECTIVE: We aimed to build a dose-response modeling framework that continuously extends the curve into the low-dose region via a quantitative integration of MOA information and to estimate MOA-based points of departure (PODs) for nonmutagenic carcinogens. METHODS: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as an example to demonstrate the new dose-response modeling framework. There were three major steps included: a) identifying and extracting key quantifiable events (KQEs), b) calculating essential doses that sequentially activate KQEs using the benchmark dose (BMD) methodology, and c) characterizing pathway dose-response relationship for MOA-based POD estimation. RESULTS: We identified and extracted six KQEs and corresponding essential events composing the MOA of TCDD-induced liver tumors. With the essential doses estimated from the BMD method using various settings, three link functions were applied to model the pathway dose-response relationship. Given a toxicologically plausible definition of adversity, an MOA-based POD was derived from the pathway dose-response curve. The estimated MOA-based PODs were generally comparable with traditional PODs and can be further used to calculate reference doses (RfDs). CONCLUSIONS: The proposed framework quantitatively integrated mechanistic information in the modeling process and provided a promising strategy to harmonize cancer and noncancer dose-response assessment through pathway dose-response modeling. However, the framework can also be limited by data availability and the understanding of the underlying mechanism. https://doi.org/10.1289/EHP12677.

A mode of action-based probabilistic framework of dose-response assessment for nonmutagenic liver carcinogens: a case study of PCB-126.

A main function of dose-response assessment is to estimate a "safe" dose in the target population to support chemical risk assessment. Typically, a "safe" dose is developed differently for cancer and noncancer effects based on a 2-step procedure, ie, point of departure (POD) derivation and low-dose extrapolation. However, the current dose-response assessment framework is criticized for its dichotomized strategy without integrating the mode of action (MOA) information. The objective of this study was, based on our previous work, to develop a MOA-based probabilistic dose-response framework that quantitatively synthesizes a biological pathway in a dose-response modeling process to estimate the risk of chemicals that have carcinogenic potential. 3,3',4,4',5-Pentachlorobiphenyl (PCB-126) was exemplified to demonstrate our proposed approach. There were 4 major steps in the new modeling framework, including (1) key quantifiable events (KQEs) identification and extraction, (2) essential dose calculation, (3) MOA-based POD derivation, and (4) MOA-based probabilistic reference dose (RfD) estimation. Compared with reported PODs and traditional RfDs, the MOA-based estimates derived from our approach were comparable and plausible. One key feature of our approach was the use of overall MOA information to build the dose-response relationship on the entire dose continuum including the low-dose region. On the other hand, by adjusting uncertainty and variability in a probabilistic manner, the MOA-based probabilistic RfDs can provide useful insights of health protection for the specific proportion of population. Moreover, the proposed framework had important potential to be generalized to assess different types of chemicals other than nonmutagenic carcinogens, highlighting its utility to improve current chemical risk assessment.

Evaluation of the carcinogenicity of carbon tetrachloride.

Carbon tetrachloride (CCl4) has been extensively used and reported to produce toxicity, most notably involving the liver. Carbon tetrachloride metabolism involves CYP450-mediated bioactivation to trichloromethyl and trichloromethyl peroxy radicals, which are capable of macromolecular interaction with cell components including lipids and proteins. Radical interaction with lipids produces lipid peroxidation which can mediate cellular damage leading to cell death. Chronic exposure with CCl4 a rodent hepatic carcinogen with a mode of action (MOA) exhibits the following key events: 1) metabolic activation; 2) hepatocellular toxicity and cell death; 3) consequent regenerative increased cell proliferation; and 4) hepatocellular proliferative lesions (foci, adenomas, carcinomas). The induction of rodent hepatic tumors is dependent upon the dose (concentration and exposure duration) of CCl4, with tumors only occurring at cytotoxic exposure levels. Adrenal benign pheochromocytomas were also increased in mice at high CCl4 exposures; however, these tumors are not of relevant importance to human cancer risk. Few epidemiology studies that have been performed on CCl4, do not provide credible evidence of enhanced risk of occurrence of liver or adrenal cancers, but these studies have serious flaws limiting their usefulness for risk assessment. This manuscript summarizes the toxicity and carcinogenicity attributed to CCl4, specifically addressing MOA, dose-response, and human relevance.

miR-34a regulates macrophage-associated inflammation and angiogenesis in alcohol-induced liver injury.

BACKGROUND: Alcohol-associated liver disease (ALD) is a syndrome of progressive inflammatory liver injury and vascular remodeling associated with long-term heavy intake of ethanol. Elevated miR-34a expression, macrophage activation, and liver angiogenesis in ALD and their correlation with the degree of inflammation and fibrosis have been reported. The current study aims to characterize the functional role of miR-34a-regulated macrophage- associated angiogenesis during ALD. METHODS RESULTS: We identified that knockout of miR-34a in 5 weeks of ethanol-fed mice significantly decreased the total liver histopathology score and miR-34a expression, along with the inhibited liver inflammation and angiogenesis by reduced macrophage infiltration and CD31/VEGF-A expression. Treatment of murine macrophages (RAW 264.7) with lipopolysaccharide (20 ng/mL) for 24 h significantly increased miR-34a expression, along with the enhanced M1/M2 phenotype changes and reduced Sirt1 expression. Silencing of miR-34a significantly increased oxygen consumption rate (OCR) in ethanol treated macrophages, and decreased lipopolysaccharide-induced activation of M1 phenotypes in cultured macrophages by upregulation of Sirt1. Furthermore, the expressions of miR-34a and its target Sirt1, macrophage polarization, and angiogenic phenotypes were significantly altered in isolated macrophages from ethanol-fed mouse liver specimens compared to controls. TLR4/miR-34a knockout mice and miR-34a Morpho/AS treated mice displayed less sensitivity to alcohol-associated injury, along with the enhanced Sirt1 and M2 markers in isolated macrophages, as well as reduced angiogenesis and hepatic expressions of inflammation markers MPO, LY6G, CXCL1, and CXCL2. CONCLUSION: Our results show that miR-34a-mediated Sirt1 signaling in macrophages is essential for steatohepatitis and angiogenesis during alcohol-induced liver injury. These findings provide new insight into the function of microRNA-regulated liver inflammation and angiogenesis and the implications for reversing steatohepatitis with potential therapeutic benefits in human alcohol-associated liver diseases.

US regulations to curb alleged cancer causes are ineffectual and compromised by scientific, constitutional and ethical violations.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

Assessment of the mode of action underlying development of liver lesions in mice following oral exposure to HFPO-DA and relevance to humans.

HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate) is a short-chain polyfluorinated alkyl substance (PFAS) used in the manufacture of some types of fluorinated polymers. Like many PFAS, toxicity studies with HFPO-DA indicate the liver is the primary target of toxicity in rodents following oral exposure. Due to the structural diversity of PFAS, the mode of action (MOA) can differ between PFAS for the same target tissue. There is significant evidence for involvement of peroxisome proliferator-activated receptor alpha (PPARα) activation based on molecular and histopathological responses in the liver following HFPO-DA exposure, but other MOAs have also been hypothesized based on limited evidence. The MOA underlying the liver effects in mice exposed to HFPO-DA was assessed in the context of the Key Events (KEs) outlined in the MOA framework for PPARα activator-induced rodent hepatocarcinogenesis. The first 3 KEs (ie, PPARα activation, alteration of cell growth pathways, and perturbation of cell growth/survival) are supported by several lines of evidence from both in vitro and in vivo data available for HFPO-DA. In contrast, alternate MOAs, including cytotoxicity, PPARγ and mitochondrial dysfunction are generally not supported by the scientific literature. HFPO-DA-mediated liver effects in mice are not expected in humans as only KE 1, PPARα activation, is shared across species. PPARα-mediated gene expression in humans produces only a subset (ie, lipid modulating effects) of the responses observed in rodents. As such, the adverse effects observed in rodent livers should not be used as the basis of toxicity values for HFPO-DA for purposes of human health risk assessment.

A Collaborative Initiative to Establish Genomic Biomarkers for Assessing Tumorigenic Potential to Reduce Reliance on Conventional Rodent Carcinogenicity Studies.

There is growing recognition across broad sectors of the scientific community that use of genomic biomarkers has the potential to reduce the need for conventional rodent carcinogenicity studies of industrial chemicals, agrochemicals, and pharmaceuticals through a weight-of-evidence approach. These biomarkers fall into 2 major categories: (1) sets of gene transcripts that can identify distinct tumorigenic mechanisms of action; and (2) cancer driver gene mutations indicative of rapidly expanding growth-advantaged clonal cell populations. This call-to-action article describes a collaborative approach launched to develop and qualify biomarker gene expression panels that measure widely accepted molecular pathways linked to tumorigenesis and their activation levels to predict tumorigenic doses of chemicals from short-term exposures. Growing evidence suggests that application of such biomarker panels in short-term exposure rodent studies can identify both tumorigenic hazard and tumorigenic activation levels for chemical-induced carcinogenicity. In the future, this approach will be expanded to include methodologies examining mutations in key cancer driver gene mutation hotspots as biomarkers of both genotoxic and nongenotoxic chemical tumor risk. Analytical, technical, and biological validation studies of these complementary genomic tools are being undertaken by multisector and multidisciplinary collaborative teams within the Health and Environmental Sciences Institute. Success from these efforts will facilitate the transition from current heavy reliance on conventional 2-year rodent carcinogenicity studies to more rapid animal- and resource-sparing approaches for mechanism-based carcinogenicity evaluation supporting internal and regulatory decision-making.

Constitutive androstane receptor (CAR) mediates dieldrin-induced liver tumorigenesis in mouse.

Dieldrin has been shown to induce liver tumors selectively in mice. Although the exact mechanism is not fully understood, previous studies from our laboratory and others have shown that dieldrin induced liver tumors in mice through a non-genotoxic mechanism acting on tumor promotion stage. Two studies were performed to examine the role of nuclear receptor activation as a possible mode of action (MOA) for dieldrin-induced mouse liver tumors. In the initial study, male C57BL/6 mice (6- to 8-week old) were treated with dieldrin in diet (10 ppm) for 7, 14, and 28 days. Phenobarbital (PB), beta-naphthoflavone (BNF) and Di (2-ethylhexyl) phthalate (DEHP) were included as positive controls in this study for evaluating the involvement of CAR (constitutive androstane receptor), AhR (aryl hydrocarbon receptor) or PPARα (peroxisome proliferator activated receptor alpha) in the MOA of dieldrin hepatocarcinogenesis. A significant increase in hepatocyte DNA synthesis (BrdU incorporation) was seen in treated mice compared with the untreated controls. Analysis of the expression of the nuclear receptor responsive genes revealed that dieldrin induced a significant increase in the expression of genes specific to CAR activation (Cyp2b10, up to 400- to 2700-fold) and PXR activation (Cyp3a11, up to 5- to 11-fold) over untreated controls. The AhR target genes Cyp1a1 and Cyp1a2 were also slightly induced (2.0- to 3.7-fold and 1.7- to 2.8-fold, respectively). PPARα activation was not seen in the liver following dieldrin treatment. In addition, consistent with previous studies in our lab, treatment with dieldrin produced significant elevation in the hepatic oxidative stress. In a subsequent study using CAR, PXR, and CAR/PXR knockout mice, we confirmed that the dieldrin-induced liver effects in mouse were only mediated by the activation of CAR receptor. Based on these findings, we propose that dieldrin induced liver tumors in mice through a nuclear receptor CAR-mediated mode of action. The previously observed oxidative stress/damage may be an associated or modifying factor in the process of dieldrin-induced liver tumor formation subsequent to the CAR activation.

A toxicogenomic approach for the risk assessment of the food contaminant acetamide.

Acetamide (CAS 60-35-5) is detected in common foods. Chronic rodent bioassays led to its classification as a group 2B possible human carcinogen due to the induction of liver tumors in rats. We used a toxicogenomics approach in Wistar rats gavaged daily for 7 or 28 days at doses of 300 to 1500 mg/kg/day (mkd) to determine a point of departure (POD) and investigate its mode of action (MoA). Ki67 labeling was increased at doses ≥750 mkd up to 3.3-fold representing the most sensitive apical endpoint. Differential gene expression analysis by RNA-Seq identified 1110 and 1814 differentially expressed genes in male and female rats, respectively, following 28 days of treatment. Down-regulated genes were associated with lipid metabolism while up-regulated genes included cell signaling, immune response, and cell cycle functions. Benchmark dose (BMD) modeling of the Ki67 labeling index determined the BMD10 lower confidence limit (BMDL10) as 190 mkd. Transcriptional BMD modeling revealed excellent concordance between transcriptional POD and apical endpoints. Collectively, these results indicate that acetamide is most likely acting through a mitogenic MoA, though specific key initiating molecular events could not be elucidated. A POD value of 190 mkd determined for cell proliferation is suggested for risk assessment purposes.

Icariin protects rotenone-induced neurotoxicity through induction of SIRT3.

Sirtuin-3 (SIRT3) is a mitochondrial NAD + -dependent deacetylase that is essential in regulating mitochondrial proteins and maintaining cellular antioxidant properties. It has been reported that icariin (ICA) is neuroprotective over various neurotoxicant induced oxidative stress. This study aimed to determine whether ICA exerts neuroprotective effects on rotenone (ROT)-induced neurotoxicity through activation of SIRT3. Rats treated with ROT exhibited a marked loss of dopamine (DA) neurons and a decline in motor function, along with a decrease in protein expressions of SIRT3 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the substantia nigra (SN). Administration of ICA significantly alleviated the loss of DA neurons, improved behavioral function, and concomitantly enhanced SIRT3 and PGC-1α expressions. The neuroprotective effect of ICA on ROT-induced cytotoxicity was further confirmed in the PC12 cell model, which showed significant improvement in the survival of ROT-treated cells with ICA pretreatment. The cytoprotective effect of ICA was abolished in ROT-treated cells by SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP), along with a resultant decrease in PGC-1α expression. In addition, knockdown of PGC-1α by siRNA suppressed ICA-mediated protective effects but did not affect SIRT3 expression, indicating the role of regulation of PGC-1α by SIRT3 in the protective action of ICA. Furthermore, we showed that ICA improved mitochondrial respiration, oxidative status, enhanced antioxidant enzyme SOD activity and GSH/GSSG ratio in cells treated with ROT. However, these protective effects of ICA on ROT-treated cells was markedly abolished by SIRT3 inhibitor 3-TYP. Our findings demonstrate that ICA exerts a neuroprotective role through upregulation of SIRT3.

Modulation of xenobiotic nuclear receptors in high-fat diet induced non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the western countries. The histological spectrum of NAFLD includes simple steatosis, steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. Nuclear receptors are a large group of ligand-dependent transcription factors that sense the environmental and endogenous changes and regulate numerous physiological and pathological processes. Accumulating evidence has suggested that a dysregulation of nuclear receptors in NAFLD may effect on the metabolism of endogenous and exogenous chemicals in the liver. The current study was designed to systematically characterize the time-dependent modulation of nuclear receptors including the peroxisome proliferator-activated receptors (PPARs), the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), the liver X receptor (LXR), and the farnesoid X receptor (FXR) in the progression of NAFLD. Male C57BL/6 mice fed by a high fat diet were used to induce NAFLD. Hepatic steatosis, lobular inflammation, progressive fibrosis, increased hepatocyte DNA synthesis, and liver tumor formation were observed at various time points in our mouse model. During the development of hepatic steatosis (8-16 weeks), PPARα was activated as indicated by its target genes as well as the elevated peroxisomal acyl-CoA oxidase activity. The mRNA level of Pparγ was also upregulated while Pparδ gene expression was significantly reduced during the development of hepatic steatosis. PXR target gene Cyp3a11 was consistently increased 3-4-fold in addition to the increased microsomal Cyp3a enzymatic activity at all stages of NAFLD. In contrast, CAR mediated Cyp2b10 gene expression was found increased only by week 12. LXRα target genes Abcg5 and Abcg8 were significantly elevated during the whole course of NAFLD. The mRNA of Fxr was downregulated at 24 and 32 weeks in high fat diet fed mice, which might correlate with the development of progressive fibrosis at the stage of steatohepatitis. The results of our study provided a systematic evaluation of the changes of nuclear receptors and their downstream chemicalmetabolism and transport enzymes in the progression of NAFLD.

Effect of polyhexamethylene biguanide on rat liver.

Polyhexamethylene biguanide (PHMB), an amphiphilic polymeric biocide, increased liver tumor incidence in male and female rats at 1000 and 1500 mg/L in drinking water, but not at 500 mg/L in previous studies. In another study, PHMB administered in diet at 4000 mg/kg was negative for hepatocellular tumors. The present studies evaluated bioavailability and distribution of PHMB administered in drinking water and diet and possible modes of action (MOA). PHMB in drinking water was unpalatable during the first 3 days, resulting in markedly decreased food consumption and decreased body weight. Ki-67 labeling index was increased in hepatocytes and endothelial cells dose responsively with PHMB administered in drinking water but not diet. Vitamin E had no effect on this. There was no cytotoxicity by histopathology or serum enzymes, and no increase in cytokines TNFα, IL-1α or NF-κB. Focal iron deposition in sinusoidal lining cells was detected. Microarray analyses were non-contributory. No effect on CAR or PPARα activation was detected. 14C-PHMB administered at 500, 1000, or 1500 mg/L in the drinking water or 4000 mg/kg in the diet was nearly completely absorbed and excreted in urine, with some fecal excretion. The hypothesized MOA for liver tumors induced by PHMB in drinking water is: 1) severe dehydration and starvation because of unpalatability, followed by ingestion with rapid absorption and urinary excretion; 2) increased hepatocyte proliferation; and 3) induction of hepatocellular foci and tumors. The PHMB-induced rat hepatocellular tumors are unlikely to pose a human cancer risk. However, the actual MOA has not been determined.

Oxidative Stress and Cancer.

BACKGROUND: Cancer is considered a major cause of death worldwide. The etiology of cancer is linked to environmental and genetic inheritance causes. Approximately 90 percent of all human cancers have an environmental cause (non-genetic inheritance) predominantly through lifestyle choices (smoking, diet, UV radiation) while the remaining due to infections and chemical exposure. Cancer is a multistage process that involves mutational changes and uncontrolled cell proliferation. Research has firmly established a causal and contributory role of oxidative stress and oxidative damage in cancer initiation and progression. METHODS: The purpose of this article is to review the role that oxidative stress and reactive oxygen species play in the development of cancer. Both endogenous and exogenous sources of reactive oxygen species result in increased oxidative stress in the cell. Excess reactive oxygen fumed can result in damage to and modification of cellular macromolecules most importantly genomic DNA that can produce mutations. In addition, oxidative stress modulates gene expression of downstream targets involved in DNA repair, cell proliferation and antioxidants. The modulation of gene expression by oxidative stress occurs in part through activation or inhibition of transcription factors and second messengers. The role of single nuclear polymorphism for oxidative DNA repair and enzymatic antioxidants is important in determining the potential human cancer risk. CONCLUSION: oxidative stress and the resulting oxidative damage are important contributors to the formation and progression of cancer.

The PPARα-dependent rodent liver tumor response is not relevant to humans: addressing misconceptions.

A number of industrial chemicals and therapeutic agents cause liver tumors in rats and mice by activating the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). The molecular and cellular events by which PPARα activators induce rodent hepatocarcinogenesis have been extensively studied elucidating a number of consistent mechanistic changes linked to the increased incidence of liver neoplasms. The weight of evidence relevant to the hypothesized mode of action (MOA) for PPARα activator-induced rodent hepatocarcinogenesis is summarized here. Chemical-specific and mechanistic data support concordance of temporal and dose-response relationships for the key events associated with many PPARα activators. The key events (KE) identified in the MOA are PPARα activation (KE1), alteration in cell growth pathways (KE2), perturbation of hepatocyte growth and survival (KE3), and selective clonal expansion of preneoplastic foci cells (KE4), which leads to the apical event-increases in hepatocellular adenomas and carcinomas (KE5). In addition, a number of concurrent molecular and cellular events have been classified as modulating factors, because they potentially alter the ability of PPARα activators to increase rodent liver cancer while not being key events themselves. These modulating factors include increases in oxidative stress and activation of NF-kB. PPARα activators are unlikely to induce liver tumors in humans due to biological differences in the response of KEs downstream of PPARα activation. This conclusion is based on minimal or no effects observed on cell growth pathways and hepatocellular proliferation in human primary hepatocytes and absence of alteration in growth pathways, hepatocyte proliferation, and tumors in the livers of species (hamsters, guinea pigs and cynomolgus monkeys) that are more appropriate human surrogates than mice and rats at overlapping dose levels. Despite this overwhelming body of evidence and almost universal acceptance of the PPARα MOA and lack of human relevance, several reviews have selectively focused on specific studies that, as discussed, contradict the consensus opinion and suggest uncertainty. In the present review, we systematically address these most germane suggested weaknesses of the PPARα MOA.

Autophagy plays a protective role in Mn-induced toxicity in PC12 cells.

Excessive environmental or occupational exposure to manganese (Mn) is associated with increased risk of neuron degenerative disorders. Oxidative stress and mitochondrial dysfunction are the main mechanisms of Mn mediated neurotoxicity. Selective removal of damaged mitochondria by autophagy has been proposed as a protective mechanism against neuronal toxicant-induced neurotoxicity. Whether autophagic flux plays a role in Mn-induced cytotoxicity remains to be fully elucidated. The present study was designed to investigate the effect of Mn exposure on autophagy, and how modulation of autophagic flux alters the sensitivities of cells to Mn-elicited cytotoxicity. Rat adrenal pheochromocytoma PC12 cells were treated with Mn for 24h to establish a cellular mode of Mn toxicity. Treatment of cells with Mn resulted in increased expression of autophagic marker LC3-II protein, as well as accumulation of p62, indicating an interference of autophagy flux caused by Mn. Pre-incubation of cells with antioxidant N-acetyl-l-cysteine (NAC) or resveratrol improved cell survival, accompanied by decreased LC3-II expression and increased expression level of p62, suggesting a down regulation of autophagy flux. To further determine the role of autophagy in Mn-induced cytotoxicity, the effect of chloroquine and rapamycin on cell viability was examined. Inhibition of autophagy flux by chloroquine exacerbated Mn-induced cytotoxicity, while induction of autophagy by rapamycin significantly reduced cell death caused by Mn. Furthermore, it was found that rapamycin, NAC and resveratrol improved cellular oxygen consumption accompanied by a decrease in cellular ROS generation and increase in GSH level, while chloroquine suppressed cellular respiration and deteriorated cellular oxidative stress. Collectively, these results demonstrate that autophagy plays a protective role in Mn-induced cell toxicity. Antioxidants NAC and resveratrol confer protective role in Mn toxicity mainly through maintaining mitochondrial dynamics and function, other than a modulation of autophagy flux.