Liver-specific deletion of microRNA-34a alleviates ductular reaction and liver fibrosis during experimental cholestasis.

Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by inflammatory responses and fibrotic scar formation leading to cholestasis. Ductular reaction and liver fibrosis are typical liver changes seen in human PSC and cholestasis patients. The current study aimed to clarify the role of liver-specific microRNA-34a in the cholestasis-associated ductular reaction and liver fibrosis. We demonstrated that miR-34a expression was significantly increased in human PSC livers along with the enhanced ductular reaction, cellular senescence, and liver fibrosis. A liver-specific miR-34a knockout mouse was established by crossing floxed miR-34a mice with albumin-promoter-driven Cre mice. Bile duct ligation (BDL) induced liver injury characterized by necrosis, fibrosis, and immune cell infiltration. In contrast, liver-specific miR-34a knockout in BDL mice resulted in decreased biliary ductular pathology associated with the reduced cholangiocyte senescence and fibrotic responses. The miR-34a-mediated ductular reactions may be functioning through Sirt-1-mediated senescence and fibrosis. The hepatocyte-derived conditioned medium promoted LPS-induced fibrotic responses and senescence in cholangiocytes, and miR-34a inhibitor suppressed these effects, further supporting the involvement of paracrine regulation. In conclusion, we demonstrated that liver-specific miR-34a plays an important role in ductular reaction and fibrotic responses in a BDL mouse model of cholestatic liver disease.

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.

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.

Dysbiosis in gastrointestinal pathophysiology: Role of the gut microbiome in Gulf War Illness.

Gulf War Illness (GWI) has been reported in 25%-35% of veterans returned from the Gulf war. Symptoms of GWI are varied and include both neurological and gastrointestinal symptoms as well as chronic fatigue. Development of GWI has been associated with chemical exposure particularly with exposure to pyridostigmine bromide (PB) and permethrin. Recent studies have found that the pathology of GWI is connected to changes in the gut microbiota, that is the gut dysbiosis. In studies using animal models, the exposure to PB and permethrin resulted in similar changes in the gut microbiome as these found in GW veterans with GWI. Studies using animal models have also shown that phytochemicals like curcumin are beneficial in reducing the symptoms and that the extracellular vesicles (EV) released from gut bacteria and from the intestinal epithelium can both promote diseases and suppress diseases through the intercellular communication mechanisms. The intestinal epithelium cells produce EVs and these EVs of intestinal epithelium origin are found to suppress inflammatory bowel disease severity, suggesting the benefits of utilizing EV in treatments. On the contrary, EV from the plasma of septic mice enhanced the level of proinflammatory cytokines in vitro and neutrophils and macrophages in vivo, suggesting differences in the EV depending on the types of cells they were originated and/or influences of environmental changes. These studies suggest that targeting the EV that specifically have positive influences may become a new therapeutic strategy in the treatment of veterans with GWI.

Endothelial dysfunction in pathological processes of chronic liver disease during aging.

Aging is associated with gradual changes in liver structure and physiological/pathological functions in hepatic cells including hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells (HSCs), and liver sinusoidal endothelial cells (LSECs). LSECs are specialized hepatic endothelial cells that regulate liver homeostasis. These cells actively impact the hepatic microenvironment as they have fenestrations and a thin morphology to allow substance exchange between circulating blood and the liver tissue. As aging occurs, LSECs have a reduction in both the number and size of fenestrations, which is referred to as pseudocapillarization. This along with the aging of the liver leads to increased oxidative stress, decreased availability of nitric oxide, decreased hepatic blood flow, and increased inflammatory cytokines in LSECs. Vascular aging can also lead to hepatic hypoxia, HSC activation, and liver fibrosis. In this review, we described the basic structure of LSECs, and the effect of LSECs on hepatic inflammation and fibrosis during aging process. We briefly summarized the changes of hepatic microcirculation during liver inflammation, the effect of aging on the clearance function of LSECs, the interactions between LSECs and immunity, hepatocytes or other hepatic nonparenchymal cells, and the therapeutic intervention of liver diseases by targeting LSECs and vascular system. Since LSECs play an important role in the development of liver fibrosis and the changes of LSEC phenotype occur in the early stage of liver fibrosis, the study of LSECs in the fibrotic liver is valuable for the detection of early liver fibrosis and the early intervention of fibrotic response.

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.

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.

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.

Assessment of the Mode of Action Underlying the Effects of GenX in Mouse Liver and Implications for Assessing Human Health Risks.

GenX is an alternative to environmentally persistent long-chain perfluoroalkyl and polyfluoroalkyl substances. Mice exposed to GenX exhibit liver hypertrophy, elevated peroxisomal enzyme activity, and other apical endpoints consistent with peroxisome proliferators. To investigate the potential role of peroxisome proliferator-activated receptor alpha (PPARα) activation in mice, and other molecular signals potentially related to observed liver changes, RNA sequencing was conducted on paraffin-embedded liver sections from a 90-day subchronic toxicity study of GenX conducted in mice. Differentially expressed genes were identified for each treatment group, and gene set enrichment analysis was conducted using gene sets that represent biological processes and known canonical pathways. Peroxisome signaling and fatty acid metabolism were among the most significantly enriched gene sets in both sexes at 0.5 and 5 mg/kg GenX; no pathways were enriched at 0.1 mg/kg. Gene sets specific to the PPARα subtype were significantly enriched. These findings were phenotypically anchored to histopathological changes in the same tissue blocks: hypertrophy, mitoses, and apoptosis. In vitro PPARα transactivation assays indicated that GenX activates mouse PPARα. These results indicate that the liver changes observed in GenX-treated mice occur via a mode of action (MOA) involving PPARα, an important finding for human health risk assessment as this MOA has limited relevance to humans.

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 simple automated method for continuous fieldwise measurement of microvascular hemodynamics.

Microvascular perfusion dynamics are vital to physiological function and are frequently dysregulated in injury and disease. Typically studies measure microvascular flow in a few selected vascular segments over limited time, failing to capture spatial and temporal variability. To quantify microvascular flow in a more complete and unbiased way we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow), a macro for FIJI open-source image analysis software. Using high-speed microvascular flow movies, STAFF generates kymographs for every time interval for every vascular segment, calculates flow velocities from red blood cell shadow angles, and outputs the data as color-coded velocity map movies and spreadsheets. In untreated mice, analyses demonstrated profound variation even between adjacent sinusoids over seconds. In acetaminophen-treated mice we detected flow reduction localized to pericentral regions. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of microvascular flow.

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.

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.

Human relevance of rodent liver tumors: Key insights from a Toxicology Forum workshop on nongenotoxic modes of action.

The Toxicology Forum sponsored a workshop in October 2016, on the human relevance of rodent liver tumors occurring via nongenotoxic modes of action (MOAs). The workshop focused on two nuclear receptor-mediated MOAs (Constitutive Androstane Receptor (CAR) and Peroxisome Proliferator Activated Receptor-alpha (PPARα), and on cytotoxicity. The goal of the meeting was to review the state of the science to (1) identify areas of consensus and differences, data gaps and research needs; (2) identify reasons for inconsistencies in current regulatory positions; and (3) consider what data are needed to demonstrate a specific MOA, and when additional research is needed to rule out alternative possibilities. Implications for quantitative risk assessment approaches were discussed, as were implications of not considering MOA and dose in hazard characterization and labeling schemes. Most, but not all, participants considered the CAR and PPARα MOAs as not relevant to humans based on quantitative and qualitative differences. In contrast, cytotoxicity is clearly relevant to humans, but a threshold applies. Questions remain for all three MOAs concerning what data are necessary to determine the MOA and to what extent it is necessary to exclude other MOAs.

Obfuscating transparency?

Several recent and prominent articles in Science and Nature deliberately mischaracterized the nature of genuine scientific evidence. Those articles take issue with the United States Environmental Protection Agency's recent proposal to structure its policies and rules only from studies with transparently published raw data. The articles claim it is an effort to obfuscate with transparency, by eliminating a host of studies not offering raw data. A remarkable declaration by a Science editorial is that properly trained experts can verify the scientific evidence of studies without access to raw data, We assert the Agency's proposal must be sustained. Transparency in reporting is a fundamental ethical imperative of objective scientific research justifying massive official regulations and policies. Putative hazards bereft of independent scientific evidence will continue to stoke public anxieties, calling for precautionary regulations and policies. These should rely not on spurious science but on transparent tradeoffs between the smallest exposures compatible with utility and with social perceptions of affordable precaution.

Investigation of the mechanism of triclosan induced mouse liver tumors.

Chronic dietary exposure to Triclosan (TCS) produced increased incidence of liver tumors in mice. The mechanism for liver tumor induction has been attributed to activation of either peroxisome proliferator activated receptor α (PPARα) or constitutive androstane receptor (CAR). To further define the mechanism of TCS induced liver tumors, male CD-1 and C57BL/6 mice were treated with TCS at 0, 10, 100 and 200 mg/kg diet/day for 14 or 28 days. In addition, a recovery group and positive control groups for CAR or PPARα activation with either phenobarbital or diethylhexyl-phthalate were included in the 14-day study. TCS induced a dose-dependent increase in relative liver weight and centrilobular hypertrophy in both strains of mice. Hepatocyte DNA synthesis (BrdU labeling) was also increased in a dose-related pattern. In comparison with previous studies, TCS induced a significant increase in CAR/PXR (Cyp2b10, Cyp3a11) and PPARα (Cyp4a10) responsive genes in both CD-1 and C57BL/6 mice. The corresponding enzyme activity for CAR (7-pentoxyresorufin-O-dealkylase) and PPARα (peroxisomal Acyl-CoA oxidase) were also significantly increased in a similar fashion. Oxidative stress related genes Gpx1 and Aox1 were increased in the C57BL/6 but not in CD-1 mice. The increases in gene expression and enzyme activities returned to control levels after 14-day recovery. The present results demonstrate that both CAR and PPARα activation are involved in the TCS induced mouse liver tumor.

Toxaphene-induced mouse liver tumorigenesis is mediated by the constitutive androstane receptor.

Toxaphene was shown to increase liver tumor incidence in B6C3F1 mice following chronic dietary exposure. Preliminary evidence supported a role for the constitutive androstane receptor (CAR) in the mode of action of toxaphene-induced mouse liver tumors. However, these results could not rule out a role for the pregnane X receptor (PXR) in liver tumor formation. To define further the nuclear receptors involved in this study, we utilized CAR, PXR and PXR/CAR knockout mice (CAR-/- , PXR-/- and PXR-/- /CAR-/- ) along with the wild-type C57BL/6. In this study CAR-responsive genes Cyp3a11 and Cyp2b10 were induced in the liver of C57BL/6 (wild-type) mice by toxaphene (30-570-fold) (at the carcinogenic dose 320 ppm) and phenobarbital (positive control) (16-420-fold) following 14 days' dietary treatment. In contrast, in CAR-/- mice, no induction of these genes was seen following treatment with either chemical. Cyp3a11 and Cyp2b10 were also induced in PXR-/- mice with toxaphene and phenobarbital but were not changed in treated PXR-/- /CAR-/- mice. Similarly, induction of liver pentoxyresorufin-O-deethylase (CAR activation) activity by toxaphene and phenobarbital was absent in CAR-/- and PXR-/- /CAR-/- mice treated with phenobarbital or toxaphene. Ethoxyresorufin-O-deethylase (EROD, represents aryl hydrocarbon receptor activation) activity in CAR-/- mice treated with toxaphene or phenobarbital was increased compared with untreated control, but lower overall in activity in comparison to the wild-type mouse. Liver EROD activity was also induced by both phenobarbital and toxaphene in the PXR-/- mice but not in the PXR-/- /CAR-/- mice. Toxaphene treatment increased 7-benzyloxyquinoline activity (a marker for PXR activation) in a similar pattern to that seen with pentoxyresorufin-O-deethylase. These observations indicate that EROD and PXR activation are evidence, as expected, of secondary overlap to primary CAR receptor activation. Together, these results definitively show that activation of the CAR nuclear receptor is the mode of action of toxaphene-induced mouse liver tumors. Copyright © 2017 John Wiley & Sons, Ltd.

Toxicology of decamethylcyclopentasiloxane (D5).

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane used in the formulation of consumer products as well as an industrial intermediate. A summary of the previous studies on the toxicology of D5 is provided. Toxicokinetic studies with D5 after dermal administration demonstrate a very low uptake of due to rapid evaporation. Following inhalation exposure, exhalation of unchanged D5 and excretion of metabolites with urine are major pathways for clearance in mammals. Due to this rapid clearance by exhalation, the potential for bioaccumulation of D5 is considered unlikely. The available toxicity data on D5 adequately cover the relevant endpoints regarding potential human health hazards. D5 was not DNA reactive or mutagenic in standard in vitro and in vivo test systems. D5 also did not induce developmental and reproductive toxicity in appropriately performed studies. In repeated studies in rats with subacute, subchronic and chronic inhalation exposure, mild effects on the respiratory tract typically seen after inhalation of irritating materials, increases in liver weight (28- and 90-day inhalation studies), and a small increase in the incidence of uterine adenocarcinoma (uterine tumor) in female rats (two-year inhalation chronic bioassay) were observed. The liver effects induced by D5 were consistent with D5 as a weak "phenobarbital-like" inducer of xenobiotic metabolizing enzymes and these effects are considered to be an adaptive response. Mechanistic studies to elucidate the mode-of-action for uterine tumor induction suggest an interaction of D5 with dopamine signal transduction pathways altering the pituitary control of the estrus cycle. The resulting estrogen imbalance may cause the small increase in uterine tumor incidence at the highest D5-exposure concentration over that seen in control rats. A genotoxic mechanism or a direct endocrine activity of D5 is not supported as a mode-of-action to account for the induction of uterine tumors by the available data.

Biological relevance of decamethylcyclopentasiloxane (D5) induced rat uterine endometrial adenocarcinoma tumorigenesis: Mode of action and relevance to humans.

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane used in the production and formulation of consumer products with potential exposure to manufacturing workers, consumer, and the general public. Following a combined 2-year inhalation chronic bioassay performed in Fischer 344 (F344) rats, an increase in uterine endometrial adenocarcinomas was noted at the highest concentration to which animals were exposed. No other neoplasms were detected. In this study, a dose of 160 ppm produced an incidence of 8% endometrial adenocarcinomas. Based on a number of experimental studies with D5, the current manuscript examines the biological relevance and possible modes of action for the uterine endometrial adenocarcinomas observed in the rat following chronic exposure to D5. Variable rates of spontaneous uterine endometrial adenocarcinomas have been reported for untreated F344 CrlBr rats. As such, we concluded that the slight increase in uterine endometrial adenocarcinomas observed in the D5 chronic bioassay might not be the result of D5 exposure but may be related to variability of the spontaneous tumor incidence in this strain of rat. However, if the uterine endometrial adenocarcinomas are related to D5-exposure, alteration in the estrous cycle in the aging F344 rat is the most likely mode of action. D5 is not genotoxic or estrogenic. The alteration in the estrous cycle is caused by a decrease in progesterone with an increase in the estrogen:progesterone ratio most likely induced by a decrease in prolactin concentration. Available data support that exposure to D5 influences prolactin concentration. Although the effects on prolactin concentrations in a number of experiments were not always consistent, the available data support the conclusion that D5 is acting via a dopamine receptor agonist-like mechanism to alter the pituitary control of the estrous cycle. In further support of this mode of action, studies in F344 aged animals showed that the effects of D5 on estrous cyclicity produced a response consistent with a dopamine-like effect and further suggest that D5 is accelerating the aging of the reproductive endocrine system in the F344 rat utilized in this study. This mode of action for uterine endometrial adenocarcinoma tumorigenesis is not relevant for humans.

Protective effects of antioxidants on acrylonitrile-induced oxidative stress in female F344 rats.

The induction of oxidative stress and damage appears to be involved in acrylonitrile induction of brain astrocytomas in rat. The present study examined the effects of dietary antioxidant supplementation on acrylonitrile-induced oxidative stress and oxidative damage in rats in vivo. To assess the effects of antioxidants on biomarkers of acrylonitrile-induced oxidative stress, female F344 rats were provided with diets containing vitamin E (0.05%), green tea polyphenols (GTP, 0.4%), N-acetyl cysteine (NAC, 0.3%), sodium selenite (0.1mg/kg), and taurine (10g/kg) for 7 days, and then co-administered with 0 and 100 ppm acrylonitrile in drinking water for 28 days. Significant increase in oxidative DNA damage in brain, evidenced by elevated 8OHdG levels, was seen in acrylonitrile-exposed rats. Supplementation with vitamin E, GTP, and NAC reduced acrylonitrile-induced oxidative DNA damage in brain while no protective effects were seen with the selenium or taurine supplementation. Acrylonitrile increased oxidative DNA damage, measured by the fpg-modified alkaline Comet assay in rat WBCs, which was reduced by supplementation of Vitamin E, GTP, NAC, selenium, and taurine. In addition to stimulation of oxidative DNA damage, acrylonitrile triggered induction of pro-inflammatory cytokines Tnfα, Il-1ß, and Ccl2, and the growth stimulatory cyclin D1 and cyclin D2 genes, which were effectively down-regulated with antioxidant treatment. Antioxidant treatment also was able to stimulate the pro-apoptotic genes Bad, Bax, and FasL and DNA repair genes Xrcc6 and Gadd45α. The results of this study support the involvement of oxidative stress in the development of acrylonitrile-induced astrocytomas and suggest that antioxidants block acrylonitrile-mediated damage through mechanisms that may involve in the suppression of inflammatory responses, inhibition of cell proliferation and stimulation of apoptosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1808-1818, 2016.