Hepatic Retinoid Stores Research Articles

Adipocyte HSL is required for maintaining circulating vitamin A and RBP4 levels during fasting

Vitamin A (retinol) is distributed via the blood bound to its specific carrier protein, retinol-binding protein 4 (RBP4). Retinol-loaded RBP4 is secreted into the circulation exclusively from hepatocytes, thereby mobilizing hepatic retinoid stores that represent the major vitamin A reserves in the body. The relevance of extrahepatic retinoid stores for circulating retinol and RBP4 levels that are usually kept within narrow physiological limits is unknown. Here, we show that fasting affects retinoid mobilization in a tissue-specific manner, and that hormone-sensitive lipase (HSL) in adipose tissue is required to maintain serum concentrations of retinol and RBP4 during fasting in mice. We found that extracellular retinol-free apo-RBP4 induces retinol release by adipocytes in an HSL-dependent manner. Consistently, global or adipocyte-specific HSL deficiency leads to an accumulation of retinoids in adipose tissue and a drop of serum retinol and RBP4 during fasting, which affects retinoid-responsive gene expression in eye and kidney and lowers renal retinoid content. These findings establish a novel crosstalk between liver and adipose tissue retinoid stores for the maintenance of systemic vitamin A homeostasis during fasting.

Transcription factor ISX mediates the cross talk between diet and immunity

The intestinal epithelium is a major site for the conversion of dietary β-carotene to retinaldehyde by the enzyme BCO1. The majority of retinaldehyde is further metabolized to retinol (vitamin A), esterified and packaged into triacylglycerol-rich chylomicrons for bodily distribution. Some serve on-site for the synthesis of retinoic acid, a hormone-like compound, which exerts pleiotropic and dominant effects on gastrointestinal immunity. We report here that the intestine-specific homeobox protein ISX is critical to control the metabolic flow of β-carotene through this important branching point of vitamin A metabolism. This transcription factor represses Bco1 gene expression in response to retinoic acid signaling. In ISX-deficient mice, uncontrolled Bco1 gene expression led to increased retinoid production in the intestine. Systemically, this production resulted in highly elevated hepatic retinoid stores. In the intestine, it increased the expression of retinoic acid-inducible target genes such as Aldh1a2, Dhrs3, and Ccr9 The β-carotene-inducible disruption of retinoid homeostasis affected gut-homing and differentiation of lymphocytes and displayed morphologically in large lymphoid follicles along the intestine. Furthermore, it was associated with an infiltration of the pancreas by gut-derived lymphocytes that manifested as a pancreatic insulitis with β-islet cell destruction and systemic glucose intolerance. Thus, our study identifies an important molecular interlink between diet and immunity and indicates that vitamin A homeostasis must be tightly controlled by ISX to maintain immunity and tolerance at the intestinal barrier.

Non-alcoholic steatohepatitis-related liver tumorigenesis is suppressed in mice lacking hepatic retinoid storage.

Non-alcoholic fatty liver disease has become one of the most common causes of chronic liver disease that can develop into a more serious form, non-alcoholic steatohepatitis, leading to liver cirrhosis and hepatocellular carcinoma. Although hepatic retinoid stores are progressively lost during the development of liver disease, how this affects steatohepatitis and its related hepatocarcinogenesis is unknown. In order to investigate these, we used subcutaneous injection of streptozotocin (0.2 mg/body) and high-fat diet to induce steatohepatitis and hepatic tumorigenesis in lecithin:retinol acyltransferase -deficient mice (n = 10), which lack stored retinoid in the liver, and control mice (n = 12). At the termination of the experiment (16 weeks of age), the development of hepatic tumors was significantly suppressed in mutant mice compared to controls. Lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in mutant mice. Mutant mice exhibited increased levels of retinoic acid-responsive genes, including p21, and decreased oxidative stress as evaluated by serum and liver markers. Our findings are consistent with the conclusion that mutant mice are less susceptible to steatohepatitis-related liver tumorigenesis due to increased retinoid signaling, which is accompanied by up-regulated p21 expression and attenuated oxidative stress.

Hepatic Detoxification of Bisphenol A is Retinoid-Dependent.

Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl) propane) is a widely used industrial chemical. The extensive distribution of BPA in the environment poses risks to humans. However, the molecular mechanisms underlying BPA toxicity as well as its effective detoxification and elimination are not well understood. We have investigated specifically for BPA the notion raised in the literature that the optimal sensing, detoxification, and elimination of xenobiotics requires retinoid (natural derivatives and synthetic analogs of vitamin A) actions. The objective of the study was to explore how retinoids, both those stored in the liver and those originating from recent oral intake, help maintain an optimal xenobiotic detoxification response, affecting mRNA expression and activities of elements of xenobiotic detoxification system upon BPA administration to mice. Wild-type and mice lacking hepatic retinoid stores (Lrat-/-) were acutely treated with BPA (50 mg/kg body weight), with or without oral supplementation with retinyl acetate. Hepatic mRNA expression levels of the genes encoding nuclear receptors and their downstream targets involved in xenobiotic biotransformation, phase I and phase II enzyme activities, and levels of oxidative damage to cellular proteins and lipids in hepatic microsomes, mitochondria and cytosol, were assessed. BPA treatment induced hepatic activities needed for its detoxification and elimination in wild-type mice. However, BPA failed to induce these activities in the livers of Lrat-/- mice. Oral supplementation with retinyl acetate restored phase I and phase II enzyme activities, but accelerated BPA-induced oxidative damage through enhancement of non-mitochondrial ROS production. Thus, the activities of the enzymes involved in the hepatic elimination of BPA require hepatic retinoid stores. The extent of hepatic damage that arises from acute BPA intoxication is directly affected by retinoid administration during the period of BPA exposure and hepatic retinoid stores that have accumulated over the lifetime of the organism.

Abstract 5240: Lacking hepatic retinoid storage in mice suppresses NASH-related hepatocarcinogenesis

Abstract Non-alcoholic fatty liver disease (NAFLD), which is strongly associated with metabolic syndrome, has become one of the most common causes of chronic liver disease in developed countries. Some patients with NAFLD develop a more serious form of the disease, non-alcoholic steatohepatitis (NASH), and NASH can develop cirrhosis or even hepatocellular carcinoma (HCC). Although hepatic retinoid (vitamin A) stores are progressively lost during the development of liver diseases, how this affects NAFLD/NASH and NASH-related hepatocarcinogenesis is unknown. In order to investigate this, we used streptozotocin (STZ) and high-fat diet (HFD) to induce NASH and related hepatic tumorigenesis in matched wild-type and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid in the liver. LRAT is the sole enzyme responsible for hepatic retinyl ester synthesis since LRAT KO mice completely lack lipid droplets in hepatic stellate cells. Mice were injected with STZ within 48 hours after birth and then fed HFD from 4 to 16 weeks of age. At the termination of the experiment, liver tumors were observed macroscopically and microscopically in both groups. In LRAT KO mice, the development of hepatic adenoma and HCC was significantly suppressed compared to control group. LRAT KO mice showed decreased expression levels of cyclin D1 and TGF-beta mRNA in the liver. The serum levels of d-ROMs and BAP were measured and the d-ROM/BAP ratio, which indicates oxidative stress, markedly decreased in LRAT KO mice. The fibrosis in the liver of the LRAT KO mice was decreased. In addition, liver fibrosis in LRAT KO mice was suppressed. These findings indicate that LRAT KO mice are less susceptible to STZ and HFD-induced liver tumorigenesis due to regulation of cell cycle and attenuation of oxidative stress. The amount of hepatic retinoid may affect the progression of NASH and the development of NASH-related HCC. Citation Format: Takayasu Ideta, Yohei Shirakami, Tsuneyuki Miyazaki, Hiroyasu Sakai, Takuji Tanaka, William S. Blaner, Masahito Shimizu. Lacking hepatic retinoid storage in mice suppresses NASH-related hepatocarcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5240.

Hepatotoxicity of bisphenol A under conditions of differential supplementation with retinoids.

Classical xenoestrogenic in vivo effects of bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) are well-described in the literature, however the molecular mechanisms of BPA-induced hepatotoxicity are not fully characterized. The work is aimed to assess biochemical markers of BPA induced hepatotoxicity under conditions of differential supplementation with retinoids. We demonstrate that the absence of hepatic retinyl esters as the main form of vitamin A storage provides for a resistance to BPA induced liver damage. Retinoid supplementation increases the hepatotoxic effects of bisphenol A, evidenced in higher indexes of oxidative damage of lipids, proteins and non-protein thiol groups as well as increase of serum alanine aminotransferase activity and myeloperoxidase activity in liver parenchyma. The absence of hepatotoxicity signs when hepatic retinoid stores are depleted and their presence during normal or excessive retinoid supplementation suggest that hepatic retinoid availability is one of the factors determining the hepatotoxicity of bisphenol A.

Effects of vitamin A deficiency in the postnatal mouse heart: role of hepatic retinoid stores.

To determine whether hepatic depletion of vitamin A (VA) stores has an effect on the postnatal heart, studies were carried out with mice lacking liver retinyl ester stores fed either a VA-sufficient (LRVAS) or VA-deficient (LRVAD) diet (to deplete circulating retinol and extrahepatic stores of retinyl esters). There were no observable differences in the weights or gross morphology of hearts from LRVAS or LRVAD mice relative to sex-matched, age-matched, and genetically matched wild-type (WT) controls fed the VAS diet (WTVAS), but changes in the transcription of functionally relevant genes were consistent with a state of VAD in LRVAS and LRVAD ventricles. In silico analysis revealed that 58/67 differentially expressed transcripts identified in a microarray screen are products of genes that have DNA retinoic acid response elements. Flow cytometric analysis revealed a significant and cell-specific increase in the number of proliferating Sca-1 cardiac progenitor cells in LRVAS animals relative to WTVAS controls. Before myocardial infarction, LRVAS and WTVAS mice had similar cardiac systolic function and structure, as measured by echocardiography, but, unexpectedly, repeat echocardiography demonstrated that LRVAS mice had less adverse remodeling by 1 wk after myocardial infarction. Overall, the results demonstrate that the adult heart is responsive to retinoids, and, most notably, reducing hepatic VA stores (while maintaining circulating levels of VA) impacts ventricular gene expression profiles, progenitor cell numbers, and response to injury.

Retinoid roles in blocking hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major health issue in many countries. The prognosis of HCC is still poor due to its high recurrence rate and its resistance to chemotherapy. Retinoids have long been known to have a connection with liver diseases, including HCC. Many experimental and clinical investigations have demonstrated associations between retinoids and hepatic disease, including the loss of retinoid activity in HCC cell lines and decreased hepatic retinoid stores and altered retinoid signaling in patients with cirrhosis and HCC. Based on these findings, preclinical and clinical investigations of retinoid effects on HCC have been undertaken. Recently, clinical trial results for the use of a synthetic retinoid, acyclic retinoid (ACR), to prevent HCC recurrence were published. In addition, extensive experimental studies on the action of retinoids in liver disease, including chronic viral hepatitis and non-alcoholic fatty liver disease (NAFLD), which lead to HCC have been performed. In the first section of this review, we will summarize the effectiveness and roles of retinoid for treating liver disease and blocking HCC. Subsequently, we will focus on ACR actions in blocking HCC.

Chronic alcohol consumption has a biphasic effect on hepatic retinoid loss.

The alcohol-induced depletion of hepatic retinoid stores correlates with the progression of liver injury; however, the mechanisms underlying alcohol's effects have not been fully elucidated. Our goal was to gain a mechanistic understanding of alcohol-induced hepatic retinoid depletion. Wild-type and mutant mice were continuously fed alcohol through Lieber-DeCarli liquid diets, with matched control animals pair fed an isocaloric alcohol-free diet to ensure equal nutrient and calorie intake between groups. A systematic analysis of tissue retinol and retinyl ester levels was performed with HPLC, complemented by gene and protein expression analyses. Our results delineated 2 phases of alcohol-induced depletion of hepatic retinoid. Initially, ∼15% of hepatic retinoid content was mobilized from the liver, causing extrahepatic tissue retinoid levels to increase. Subsequently, there was a precipitous drop in hepatic retinoid content (>60%), without further retinoid accumulation in the periphery. Follow-up studies in mutant mice revealed roles for RBP, CRBP1, and CD36 in retinoid mobilization and extrahepatic retinoid uptake, as well as a role for CYP2E1 in the catabolism of hepatic retinoid. In summary, alcohol has a biphasic effect on hepatic retinoid stores, characterized by an initial phase of rapid mobilization to extrahepatic tissues followed by extensive catabolism within the liver.

Retinoids Modulate Thioacetamide-Induced Acute Hepatotoxicity

The literature indicates that retinoids can influence the metabolism and actions of xenobiotics and conversely that xenobiotics can influence the metabolism and actions of retinoids. We were interested in understanding the degree to which hepatic retinoid stores, accumulated over a lifetime, affect xenobiotic metabolism, and actions. To investigate this, we induced liver injury through administration of the hepatotoxin thioacetamide (TAA) to chow fed wild type (WT) mice and lecithin:retinol acyltransferase-deficient (Lrat(-/-)) mice that are genetically unable to accumulate hepatic retinoid stores. Within 48 h of TAA-treatment, WT mice develop liver injury as evidenced by focal necrotic areas and increases in serum ALT activity and myeloperoxidase activity in hepatic parenchyma. Simultaneously, features of hepatic encephalopathy develop, as evidenced by a 25% increase in blood ammonia and a threefold reduction of blood glucose levels. This is accompanied by reduced hepatic glutathione, and increased thiobarbituric acid reactive substances, protein carbonyl and sulfhydryl groups, and increased cytochrome P450-catalyzed hydroxylation activity and flavin-containing monooxygenase activity in microsomes prepared from WT liver. Strikingly, none of these TAA-induced effects were observed for matched Lrat(-/-) mice. To confirm that TAA hepatotoxicity depends on retinoid availability, we administered, over 48 h, four oral doses of 3000 IU retinyl acetate each to the mice. This led to the development of hepatotoxicity in Lrat(-/-) mice that was similar in extent to that observed in WT mice. Our findings establish that endogenous hepatic retinoid stores can modulate the toxicity of TAA in mice.

Abstract 181: Hepatic retinoid signaling in mouse hepatocarcinogenesis models.

Abstract Retinoids are known to possess anti-cancer effects and a clinical trial of a synthetic retinoid for hepatocellular carcinoma (HCC) is ongoing recently in Japan. Hepatic retinoid metabolism is usually impaired during development of liver diseases. Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver diseases and retinoid stores are progressively lost from HSCs leading to hepatic fibrosis, cirrhosis, and HCC. In the present study, we employed two types of genetically-modified mice for investigating hepatic retinoid metabolism and signaling in diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis models. One is lecithin:retinol acyltransferas (LRAT) knockout (KO) mouse which lacks HSC lipid droplets and has much less hepatic retinoid stores, and the other is a transgenic mouse which expresses dominant negative form of retinoic acid receptor (dnRAR) to block retinoid signaling in the liver. Male 15 day-old mice were given intraperitoneal injections of DEN (25 mg/kg body weight). Eight-month-old DEN-treated Lrat KO mice showed lower liver tumor incidence and dnRAR transgenic mice showed higher incidence than that of respective control mice. In the acute phase after DEN injection, dnRAR transgenic mice exhibited higher hepatic levels of inflammatory cytokines. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN-bioactivation, and higher levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Our results indicate that Lrat KO mice, instead of almost no hepatic retinoid storage, are less susceptible to DEN-induced hepatocarcinogenesis due to increased hepatic retinoid signaling which is accompanied by higher expression of p21 in the liver. In conclusion, retinoid signaling plays a key role in these hepatocarcinogenesis models. Citation Format: Yohei Shirakami, William S. Blaner, Masahito Shimizu, Hisataka Moriwaki, Mitsuru Seishima. Hepatic retinoid signaling in mouse hepatocarcinogenesis models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 181. doi:10.1158/1538-7445.AM2013-181

Altered hepatic retinyl ester concentration and acyl composition in response to alcohol consumption

Retinoids (vitamin A and its metabolites) are essential micronutrients that regulate many cellular processes. Greater than 70% of the body's retinoid reserves are stored in the liver as retinyl ester (RE). Chronic alcohol consumption induces depletion of hepatic retinoid stores, and the extent of this has been correlated with advancing stages of alcoholic liver disease. The goal of this study was to analyze the mechanisms responsible for depletion of hepatic RE stores by alcohol consumption. A change in the fatty-acyl composition of RE in alcohol-fed mice was observed within two weeks after the start of alcohol consumption. Specifically, alcohol-feeding was associated with a significant decline in hepatic retinyl palmitate levels; however, total RE levels were maintained by a compensatory increase in levels of usually minor RE species, particularly retinyl oleate. Our data suggests that alcohol feeding initially stimulates a futile cycle of RE hydrolysis and synthesis, and that the change in RE acyl composition is associated with a change in the acyl composition of hepatic phosphatidylcholine. The alcohol-induced change in RE acyl composition was specific to the liver, and was not seen in lung or white adipose tissue. This shift in hepatic RE fatty acyl composition is a sensitive indicator of alcohol consumption and may be an early biomarker for events associated with the development of alcoholic liver disease.

Hepatic retinoid stores are required for normal liver regeneration

Preliminary studies of liver regeneration induced by partial hepatectomy (PHE) identified a substantial depletion of hepatic retinoid stores, by greater than 70%, in regenerating livers of wild-type C57Bl/6J mice. To understand this, we compared responses of wild-type and lecithin:retinol acyltransferase (Lrat)-deficient mice, which totally lack hepatic retinoid stores, to PHE. The Lrat-deficient livers showed delayed regeneration in the first 24 h after PHE. At 12 h after PHE, we observed significantly less mRNA expression for growth factors and cytokines implicated in regulating the priming phase of liver regeneration, specifically for Hgf and Tgfα, but not Tgfβ. Compared with wild-type mice, the changes in mRNA levels for p21 and cyclins E1, B1, and A2 mRNAs and for hepatocellular BrdU incorporation and mitoses were delayed (i.e., shifted to later times) in regenerating Lrat(-/-) livers. Concentrations of all-trans-retinoic acid were significantly lower in the livers of Lrat(-/-) mice following PHE, and this was accompanied by diminished expression of known retinoid-responsive genes. At later times after PHE, the rate of liver weight restoration for Lrat(-/-) mice was parallel to that of wild-type mice, although additional biochemical differences were observed. Thus, hepatic retinoid stores are required for maintaining expression of signaling molecules that regulate cell proliferation and differentiation immediately after hepatic injury, accounting for the delayed restoration of liver mass in Lrat(-/-) mice.

Effects of polychlorobiphenyls, polybromodiphenylethers, organochlorine pesticides and their metabolites on vitamin A status in lactating grey seals

Polychlorobiphenyls (PCBs), polybromodiphenylethers (PBDEs) and organochlorine pesticides (OCPs), such as dichlorodiphenyltrichloroethane (DDT) and hexachlorobenzene (HCB), are considered as endocrine disruptors in laboratory and wild animals. This study investigated whether these compounds and their hydroxylated metabolites (HO-PCBs and HO-PBDEs) may affect the homoeostasis of vitamin A, a dietary hormone, in the blubber and serum of twenty lactating grey seals sampled at early and late lactation on the Isle of May, Scotland. The effect of naturally produced compounds such as the methoxylated (MeO)-PBDEs was also examined. Vitamin A levels in inner blubber (37±9μg/g wet weight (ww) and 92±32μg/g ww at early and late lactation, respectively) and serum (408±143 and 390±98ng/ml at early and late lactation, respectively) appeared to be positively related to ΣPCBs, ΣPBDEs and several individual PCB and PBDE congeners in inner blubber and serum. These findings may suggest enhanced mobilisation of hepatic retinoid stores and redistribution in the blubber, a storage site for vitamin A in marine mammals. We have also reported that serum concentrations of ΣHO-PCBs and 4-OH-CB107 tended to increase with circulating vitamin A levels. Although the direction of the relationships may sometimes differ from those reported in the literature, our results are in agreement with previous findings highlighting a disruption of vitamin A homoeostasis in the blubber and bloodstream following exposure to environmental pollutants. The fact that vitamin A and PCBs appeared to share common mechanisms of mobilisation and transfer during lactation in grey seals (Debier et al., 2004; Vanden Berghe et al., 2010) may also play a role in the different relationships observed between vitamin A and lipophilic pollutants.

Abstract 5555: Diethylnitrosamine-induced hepatic injury and hepatocarcinogenesis: A role for hepatic retinoids

Abstract The liver is the main tissue site of retinoid storage in the body and the non-parenchymal hepatic stellate cell (HSC) is the major cellular site of retinoid storage within the liver. Loss of retinoid-containing lipid droplets (LDs) from HSCs is one of the first events in the development of hepatic diseases, leading progressively to liver fibrosis, cirrhosis and hepatocellular carcinoma. Although the loss of LDs is a hallmark of HSC activation, the contribution of hepatic retinoid storage to hepatic injury and hepatocarcinogenesis remains unknown. Lecithin:retinol acyltransferase (LRAT) is the sole enzyme responsible for storing hepatic retinoid and LRAT knockout (KO) mice lack liver retinoid stores. Retinol-binding protein (RBP) is the principal transport protein for retinol in the blood. RBP KO mice are unable to mobilize hepatic retinoid stores and have significantly more retinoid storage in the liver compared to wild type (WT) mice. In this study, we investigated the effects of endogenous retinoids on hepatic injury and hepatocarcinogenesis. We induced hepatic injury and HCC using diethylnitrosamine (DEN) in WT, LRAT KO and RBP KO mice. Both acute (for 24 and 48 hours after DEN administration) and long-term (for 8 months after DEN administration) studies were undertaken. After DEN injection of WT, LRAT KO, and RBP KO mice, serum levels of alanine transaminase (ALT) were significantly lower and Cyclin D1 and Ki-67 mRNA expression levels were decreased in the livers of the KOs compared to WT mice. This suggests that LRAT KO and RBP KO mice are less susceptible to DEN-induced liver injury and following compensatory proliferation. Furthermore, 8-month-old DEN-treated LRAT KO (n = 18) and RBP KO (n = 13) mice showed lower HCC incidence, fewer tumors, and smaller tumors than DEN-treated WT mice (n = 24). Five-month-old male LRAT KO and RBP KO mice showed higher expression levels of retinoic acid (RA) responsive genes, such as Cyp26A1 and RARβ, mRNA in their liver compared to WT, indicating that RA signaling is upregulated in the livers of the KOs’ mice. The expression levels of p21 mRNA in LRAT KO and RBP KO mice liver were higher than WT at several time points in both acute and long-term studies. Together these results suggest that increased RA signaling and associated higher p21 expression play important roles in the lower susceptibility of these mouse models to liver injury and hepatocarcinogenesis. We are presently investigating other possibilities that the presence/absence through which endogenous hepatic retinoids might have effects on hepatic diseases, probably by modulating several cancer signaling pathways, regulating apoptosis of hepatocytes and cancer cells, or influencing the tumor-surrounding microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5555. doi:10.1158/1538-7445.AM2011-5555

Abstract 83: A role of hepatic retinoid storage in diethynitrosamine-induced hepatic injury and hepatocarcinogenesis

Abstract The liver is the main tissue site of retinoid (vitamin A and its derivatives) storage in the body and the non-parenchymal hepatic stellate cell (HSC) is the major cellular site of retinoid storage within the liver. Lipid droplets of HSCs contain a number of bioactive metabolites including retinoids. Following liver injury, HSCs undergo a process known as activation. Loss of retinyl esters, the retinoid storage form, and lipid droplets from HSCs is one of the first events in the development of hepatic disease, leading progressively to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Although the loss of lipid droplets is a hallmark of HSC activation, whether it is a cause or consequence of the HSC activation process is unclear. Moreover, the contribution of the loss of hepatic retinoid storage to hepatic injury and hepatocarcinogenesis remains unknown. To investigate the role of hepatic retinoid storage and retinoid-containing lipid droplets in hepatic injury and hepatocarcingenesis, we studied diethynitrosamine (DEN)-induced HCC in lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack retinoid-containing lipid droplets in their HSCs, and in retinol-binding protein (RBP) KO mice, which are unable to mobilize hepatic retinoid stores. We measured a number of factors implicated in hepatic injury and cancer initiation. Using an acute hepatic injury model for investigating cancer initiation, assessment of mRNA levels by qRT-PCR of TNF alpha and TGF beta in livers of LRAT KO mice indicated that these mice were more susceptible to cancer initiation than WT mice. On the other hand RBP KO mice showed less susceptibility. LRAT KO mice showed higher levels of CYP26A1, a gene whose expression is known to be highly and directly responsive to retinoic acid level, suggesting that hepatic retinoic acid levels in LRAT KO mice were higher than that of WT, and that this may influence hepatic injury and carcinogenesis. Overall, our data suggest that hepatic retinoid storage can influence the progression of hepatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 83.

Constituents within pulp mill effluent deplete retinoid stores in white sucker and bind to rainbow trout retinoic acid receptors and retinoid X receptors.

Wild female and male white sucker (Catostomus commersoni) inhabiting an area receiving pulp mill effluent had reduced hepatic levels of retinol, didehydroretinol, retinyl esters, and didehydroretinyl esters, while vitamin E levels were unaffected. This disruption of the retinoid system led us to test methanol and dichloromethane extracts from the effluent of 11 pulp mills from across Canada for their ability to bind to rainbow trout (Oncorhynchus mykiss) retinoic acid receptors (RARs) from the gill and retinoid X receptors (RXRs) from the liver. Concentrated extracts of the final effluent from 6 of the 11 pulp mills were able to displace greater than 25% of the receptor-bound [3H]all-trans retinoic acid (RA) or [3H]9-cis RA from trout RARs and RXRs, respectively. The ability of the extracts to displace retinoic acid did not appear to be linked to the pulping or treatment processes. Moreover, extracts with the greatest activity came from thermomechanical mills, suggesting the compounds may originate from the wood furnish. In addition, extracts prepared from wood furnish (wood chips: white spruce [50%], lodgepole pine [47%], and balsam fir [3%]) from one mill were able to displace [3H]RA from the RARs and RXRs. The 4-hydroxy RA, a metabolite of RA that has been shown to be generated in greater quantities in fish exposed to P450-inducing xenobiotics, was able to displace [3H]all-trans RA from trout RARs as effectively as unlabeled all-trans RA. These results suggest that pulp mill effluent may impact the retinoid system of fish at multiple sites, either by decreasing hepatic retinoid stores or through contributing additional ligands (from the wood furnish) that can bind to RA receptors.

Stored retinoids in populations of the estuarine fish Fundulus heteroclitus indigenous to PCB-contaminated and reference sites.

Concentrations of retinoids, derivatives of vitamin A, were measured in populations of the nonmigratory estuarine fish Fundulus heteroclitus, indigenous to a reference site and a site highly contaminated with polychlorinated biphenyls (PCBs) to address the hypothesis that contaminant exposure causes depletion of stored retinoids in native fish populations. To assess differences related to chemical exposure, as well as season and diet, hepatic retinoid analyses were conducted using fish collected early and late in the natural spawning season and after laboratory holding. Though hepatic retinoid composition was generally similar among groups, hepatic retinoid concentrations differed. Laboratory-held fish had higher hepatic retinyl ester concentrations than field-collected fish. Among field-collected fish, those collected early in the spawning season had higher hepatic retinyl ester concentrations than those collected later in the spawning season. Although there was no evidence of the dramatic retinoid depletions that have been reported in highly exposed populations of other fish species, hepatic retinoid stores were consistently lower in F. heteroclitus indigenous to the highly PCB-contaminated site. These results are consistent with prior findings that fish from this contaminated site are relatively insensitive to some of the toxic effects of PCB exposures, including retinoid depletion.

Biomarkers of exposure of brown bullheads (Ameiurus nebulosus) to contaminants in the lower Great Lakes, North America

AbstractBrown bullheads (Ameiurus nebulosus) from three contaminated sites (Detroit River, Hamilton Harbour, and Black River) and two relatively clean reference sites (Old Woman Creek and Bay of Quinte) in the lower Great Lakes were monitored for several biomarkers of exposure to chemical contaminants. Bullheads from the contaminated sites had elevated hepatosomatic indices, increased ethoxyresorufin‐O‐deethylase (EROD) activity, decreased hepatic retinoid stores, and a greater incidence of hepatic neoplasms. Fluorescent aromatic compounds (FACs) in the bile were also elevated in bullheads from the Black River and Hamilton Harbour in comparison to fish from reference sites. A correlation was found between reduction of hepatic retinoid stores and induction of hepatic EROD activity in bullheads. Principal component analysis of biomarker data demonstrated that biomarker patterns in Detroit River bullheads were different from those of Black River and Hamilton Harbour bullheads. Biomarkers in fish are useful monitoring tools for detecting contamination of the aquatic environment, but interpretation of results is limited by within‐and between‐site variability in the responses, some of which may be explained by mobility of the fish. In contaminated areas, bile fluorescent aromatic compounds appear to be the best indicator of recent exposure to polynuclear aromatic hydrocarbons, whereas responses to other biochemical biomarkers (e.g., EROD and retinoids) appear to be over a longer time period.