The effect of dihydropyrazines on lipopolysaccharide-stimulated human hepatoma HepG2 cells via regulating the TLR4-MyD88-mediated NF-κB signaling pathway.

Dihydropyrazines (DHPs), including 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), are glycation products that are spontaneously generated in vivo and ingested via food. DHPs generate various radicals and reactive oxygen species (ROS), which can induce the expression of several antioxidant genes in HepG2 cells. However, detailed information on DHP-response pathways remains elusive. To address this issue, we investigated the effects of DHP-3 on the nuclear factor-κB (NF-κB) pathway, a ROS-sensitive signaling pathway. In lipopolysaccharide-stimulated (LPS-stimulated) HepG2 cells, DHP-3 decreased phosphorylation levels of inhibitor of NF-κB (IκB) and NF-κB p65, and nuclear translocation of NF-κB p65. In addition, DHP-3 reduced the expression of Toll-like receptor 4 (TLR4) and the adaptor protein myeloid differentiation primary response gene 88 (MyD88). Moreover, DHP-3 suppressed the mRNA expression of tumor necrosis factor-alpha (TNFα), and interleukin-1 beta (IL-1ß). Taken together, these results suggest that DHP-3 acts as a negative regulator of the TLR4-MyD88-mediated NF-κB signaling pathway.

Nrf2 Activation Ameliorates Hepatotoxicity Induced by a Heme Synthesis Inhibitor.

Transcription factor Nrf2 protects hepatocytes against various toxicants by upregulating cytoprotective genes. The heme synthesis inhibitor 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) leads to liver injury around the portal vein, unlike other groups of toxicants that cause hemorrhage and necrosis in the centrilobular area. To examine whether and how Nrf2 protects livers from the injury, we fed DDC to Nrf2 knockout (Nrf2KO), wild-type (WT), Keap1flox/flox (Keap1-knockdown; Keap1KD), and liver-specific Keap1 knockout (Keap1-Alb) mice, as these lines of mice exhibit stepwise increases in Nrf2 protein expression levels. Liver-specific Keap1::Nrf2 double-knockout (Keap1::Nrf2-Alb) mice were also exploited to examine the contribution of Nrf2. Two weeks after DDC feeding, Keap1-Alb mice were fully recovered from body weight loss, but the WT and Nrf2KO mice were not. The liver-to-body-weight ratio of Keap1-Alb mice was significantly larger than that of WT and Nrf2KO mice. Two indicators of hepatotoxicity, alanine aminotransferase and bilirubin in plasma, were both elevated in WT mice, but downregulated in Keap1-Alb mice after the DDC-feeding. DDC-induced porphyrin accumulation was reduced in the livers of Keap1-Alb and Keap1KD mice compared with that of WT mice. When assessed by the Nqo1 level, Nrf2 expression was further enhanced by DDC in Keap1-Alb mice, suggesting that DDC may have a Keap1 independent potential to activate Nrf2. Genetic activation of Nrf2 in Keap1-Alb mice increased the extracellular excretion of porphyrins, but contrary to our expectation, hepatic damages in Nrf2KO mice appeared to be similar to that of WT mice. Based on these observations, we conclude that Nrf2 activation protects livers against DDC-elicited hepatotoxicity.

Sarcopenia in a mice model of chronic liver disease: role of the ubiquitin-proteasome system and oxidative stress.

Sarcopenia is the loss of muscle mass and strength produced by aging or secondary to chronic diseases such as chronic liver disease (CLD). Although not all types of sarcopenia involve the same features, the most common are decreased fiber diameter and myosin heavy chain (MHC) levels, increased activity of ubiquitin-proteasome system (UPS) and reactive oxygen species (ROS). In this study, we aim to characterize the development of sarcopenia secondary to CLD induced by the hepatotoxin 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). For this purpose, four-months-old male C57BL6 mice were fed with normal diet or DDC supplemented diet for 6 weeks. Functional tests to evaluate muscle strength, mobility, and motor skills were performed in alive mice. The muscle strength in isolated gastrocnemius was also assayed via electrophysiological measurements. Morphometric measures of fibers' diameter, total and ubiquitinated protein levels of myosin heavy chain (MHC), E3 ubiquitin ligases, ROS, and oxidation-dependent modified proteins in gastrocnemius tissue were also determined. Our results demonstrated that mice fed the DDC diet developed muscle wasting as evidenced by a loss of muscle mass and decreased muscle strength. The muscles of mice fed with DDC diet have a decreased diameter of fibers and MHC levels, also as increased MuRF-1 and atrogin-1 protein levels, ROS levels, and oxidation-modified protein levels. Additionally, control and DDC mice have the same food and water intake as well as mobility. Our results demonstrate mice with CLD develop sarcopenia involving decreased levels of myofibrillar proteins, increased UPS, and oxidative stress, but not for impaired caloric intake or immobility.

FAT10 knock out mice livers fail to develop Mallory-Denk bodies in the DDC mouse model.

Mallory-Denk bodies (MDBs) are aggresomes composed of undigested ubiqutinated short lived proteins which have accumulated because of a decrease in the rate of their degradation by the 26s proteasome. The decrease in the activity of the proteasome is due to a shift in the activity of the 26s proteasome to the immunoproteasome triggered by an increase in expression of the catalytic subunits of the immunoproteasome which replaces the catalytic subunits of the 26s proteasome. This switch in the type of proteasome in liver cells is triggered by the binding of IFNγ to the IFNγ sequence response element (ISRE) located on the FAT10 promoter. To determine if either FAT10 or IFNγ are essential for the formation of MDBs we fed both IFNγ and FAT10 knock out (KO) mice DDC added to the control diet for 10weeks in order to induce MDBs. Mice fed the control diet and Wild type mice fed the DDC or control diet were compared. MDBs were located by immunofluorescent double stains using antibodies to ubiquitin to stain MDBs and FAT10 to localize the increased expression of FAT10 in MDB forming hepatocytes. We found that MDB formation occurred in the IFNγ KO mice but not in the FAT10 KO mice. Western blots showed an increase in the ubiquitin smears and decreases ß 5 (chymotrypsin-like 26S proteasome subunit) in the Wild type mice fed DDC but not in the FAT10 KO mice fed DDC. To conclude, we have demonstrated that FAT10 is essential to the induction of MDB formation in the DDC fed mice.

Disparate cellular basis of improved liver repair in beta-catenin-overexpressing mice after long-term exposure to 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

Administration of a hepatotoxic diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) induces biliary damage followed by hepatocyte injury, which is repaired through atypical ductular proliferation and oval cells and their subsequent differentiation to bile duct cells and hepatocytes. In this study, we examine whether excess ß-catenin in transgenic (TG) mice would provide any reparative advantage in response to DDC. No differences in appearance or numbers of total A6-positive oval cells were observed after DDC administration. However, an increase in A6-positive "atypical hepatocytes" in the TG livers was observed after 14 and 28 days, coinciding with an increase in proliferating cell nuclear antigen-positive hepatocytes. Intriguingly, after chronic DDC administration for 150 days, a further increase in atypical hepatocytes was evident in TG mice, with higher numbers of proliferating cell nuclear antigen-positive hepatocytes exhibiting cytoplasmic/nuclear ß-catenin and α-fetoprotein but not CK19, HNF1ß, or Trop-2. Coincidently, we observed an improvement in intrahepatic cholestasis as seen by decreases in both serum bilirubin and alkaline phosphatase levels in TG mice, indicating an overall improvement in hepatic repair. TG mice exposed to DDC for 4 weeks followed by 2 days of normal chow showed decreases in alkaline phosphatase, atypical ductular proliferation, and periportal inflammation compared with wild-type animals, verifying improved biliary repair in TG livers. Thus, we report a potential role of ß-catenin in liver repair, especially in enhancing the resolution of intrahepatic cholestasis after DDC injury.

Hepatotoxin-induced changes in the adult murine liver promote MYC-induced tumorigenesis.

BACKGROUND: Overexpression of the human c-MYC (MYC) oncogene is one of the most frequently implicated events in the pathogenesis of hepatocellular carcinoma (HCC). Previously, we have shown in a conditional transgenic mouse model that MYC overexpression is restrained from inducing mitotic cellular division and tumorigenesis in the adult liver; whereas, in marked contrast, MYC induces robust proliferation associated with the very rapid onset of tumorigenesis in embryonic and neonatal mice. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that non-genotoxic hepatotoxins induce changes in the liver cellular context associated with increased cellular proliferation and enhanced tumorigenesis. Both 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and carbon tetrachloride (CCl(4)) cooperate with MYC to greatly accelerate the onset of liver cancer in an adult host to less than 7 days versus a mean latency of onset of over 35 weeks for MYC alone. These hepatotoxin-enhanced liver tumors grossly and histologically resemble embryonic and neonatal liver tumors. Importantly, we found that MYC overexpression is only capable of inducing expression of the mitotic Cyclin B1 in embryonic/neonatal hosts or adult hosts that were treated with either carcinogen. CONCLUSION/SIGNIFICANCE: Our results suggest a model whereby oncogenes can remain latently activated, but exposure of the adult liver to hepatotoxins that promote hepatocyte proliferation can rapidly uncover their malignant potential.

Hepatic alteration of tryptophan metabolism in an acute porphyria model Its relation with gluconeogenic blockage.

This study focuses on the alterations suffered by the serotoninergic and kinurenergic routes of tryptophan (TRP) metabolism in liver, and their relation with gluconeogenic phosphoenolpyruvate-carboxykinase (PEPCK) blockage in experimental acute porphyria. This porphyria was induced in rats by a combined treatment of 2-allyl-2-isopropylacetamide (100, 250, 500 mg/kg bw) and 3,5-dietoxicarbonil 1,4-dihydrocollidine (constant 50 mg/kg bw dose). Results showed a marked dose-dependent increase of all TRP pyrrolase (TRPp) forms, active (holo, total) and inactive (apo), and a decrease in the degree of enzyme saturation by heme. Increases for holo, total, and apo-TRPp were 90, 150, and 230%, respectively, at the highest dose assayed (H). The treatment also impaired the serotoninergic route of TRP metabolism in liver, causing a decrease in serotonin level (H, 38%), and a concomitant enhancement in TRP content (H, 23%). The porphyrinogenic treatment promoted a blockage in PEPCK activity (H, 30%). This occurred in correlation to the development of porphyria, to TRPp alterations and to the production of hepatic microsomal thiobarbituric acid reactive substances. Porphyria was estimated through increases in 5-aminolevulinic acid-synthase (ALA-S) activity, ALA and porphobilinogen contents, and a decrease in ferrochelatase activity. Thus, the TRP kynurenine route was augmented whereas the serotoninergic route was reduced. PEPCK blockage could be partly attributed to quinolinate generated from TRP by the increase of TRPp activity, which would be due to the effect of porphyrinogenic drugs on TRP. The contribution of ROS to PEPCK blockage is analyzed. Likewise, the implication of these results in the control of porphyrias by glucose is discussed.

A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis.

Xenobiotics and drugs may lead to cholangiopathies and biliary fibrosis, but the underlying mechanisms are largely unknown. Therefore, we aimed to characterize the cause and consequences of hepatobiliary injury and biliary fibrosis in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice as a novel model of xenobiotic-induced cholangiopathy. Liver morphology, markers of inflammation, cell proliferation, fibrosis, bile formation, biliary porphyrin secretion, and hepatobiliary transporter expression were studied longitudinally in DDC- and control diet-fed Swiss albino mice. DDC feeding led to increased biliary porphyrin secretion and induction of vascular cell adhesion molecule, osteopontin, and tumor necrosis factor-alpha expression in bile duct epithelial cells. This was associated with a pronounced pericholangitis with a significantly increased number of CD11b-positive cells, ductular reaction, and activation of periductal myofibroblasts, leading to large duct disease and a biliary type of liver fibrosis. After 4 weeks, we constantly observed intraductal porphyrin pigment plugs. Glutathione and phospholipid excretion significantly decreased over time. Expression of Ntcp, Oatp4, and Mrp2 was significantly reduced, whereas Bsep expression remained unchanged and adaptive Mrp3 and Mrp4 expression was significantly induced. We demonstrate that DDC feeding in mice leads to i) a reactive phenotype of cholangiocytes and bile duct injury, ii) pericholangitis, periductal fibrosis, ductular reaction, and consequently portal-portal bridging, iii) down-regulation of Mrp2 and impaired glutathione excretion, and iv) segmental bile duct obstruction. This model may be valuable to investigate the mechanisms of xenobiotic-induced chronic cholangiopathies and its sequels including biliary fibrosis.

A CD133-expressing murine liver oval cell population with bilineage potential.

Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

Pharmacologic transglutaminase inhibition attenuates drug-primed liver hypertrophy but not Mallory body formation.

Mallory bodies (MBs) are characteristic of several liver disorders, and consist primarily of keratins with transglutaminase-generated keratin crosslinks. We tested the effect of the transglutaminase-2 (TG2) inhibitor KCC009 on MB formation in a mouse model fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). KCC009 decreased DDC-induced liver enlargement without affecting MB formation or extent of liver injury. TG2 protein and activity increased after DDC feeding and localized within and outside hepatocytes. KCC009 inhibited DDC-induced hepatomegaly by affecting hepatocyte cell size rather than proliferation. Hence, TG2 is a potential mediator of injury-induced hepatomegaly via modulation of hepatocyte hypertrophy, and KCC009-mediated TG2 inhibition does not affect mouse MB formation.

Response of glucose metabolism enzymes in an acute porphyria model. Role of reactive oxygen species.

Acute hepatic porphyrias are human metabolic diseases characterized by the accumulation of heme precursors, such as 5-aminolevulinic acid (ALA). The administration of glucose can prevent the symptomatology of these diseases. The aim of this work was to study the relationship between glucose metabolism disturbances and the development of experimental acute hepatic porphyria, as well as the role of reactive oxygen species (ROS) through assays on hepatic key gluconeogenic and glycogenolytic enzymes; phosphoenolpyruvate carboxykinase (PEPCK) and glycogen phosphorylase (GP), respectively. Female Wistar rats were treated with three different doses of the porphyrinogenic drug 2-allyl-2-isopropylacetamide (AIA) and with a single dose of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Thus, rats were divided into the following groups: group L (100 mg AIA + 50 mg DDC/kg body wt.); group M (250 mg AIA + 50 mg DDC/kg body wt.) and group H (500 mg AIA + 50 mg DDC/kg body wt.). The control group (group C) only received vehicles (saline solution and corn oil). Acute hepatic porphyria markers ALA-synthase (ALA-S) and ferrochelatase, heme precursors ALA and porphobilinogen (PBG), and oxidative stress markers superoxide dismutase (SOD) and catalase (CAT) were also measured in hepatic tissue. On the other hand, hepatic cytosolic protein carbonyl content, lipid peroxidation and urinary chemiluminescence were determined as in vivo oxidative damage markers. All these parameters were studied in relation to the different doses of AIA/DDC. Results showed that enzymes were affected in a drug-dose-dependent way. PEPCK activity decreased about 30% in group H with respect to groups C and L, whereas GP activity decreased 53 and 38% in group H when compared to groups C and L, respectively. On the other hand, cytosolic protein carbonyl content increased three-fold in group H with respect to group C. A marked increase in urinary chemiluminescence and a definite increase in lipid peroxidation were also detected. The activity of liver antioxidant enzyme SOD showed an induction of about 235% in group H when compared to group C, whereas CAT activity diminished due to heme depletion caused by both drugs. Based on these results, we can speculate that the alterations observed in glucose metabolism enzymes could be partly related to the damage caused by ROS on their enzymatic protein structures, suggesting that they could be also linked to the beneficial role of glucose administration in acute hepatic porphyria cases.

Comparison of murine cirrhosis models induced by hepatotoxin administration and common bile duct ligation.

AIM: To build up the research models of hepatic fibrosis in mice. METHODS: Inbred wild-type FVB/N mice were either treated with alpha-naphthyl-isothiocyanate (ANIT), allyl alcohol (AA), carbon tetrachloride (CCl(4)), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), and silica, or subjected to common bile duct ligation (CBDL) to induce hepatic injury. Liver biopsies were performed every 4 wk to evaluate hepatic fibrosis over a period of 6 mo. Cumulative cirrhosis and survival curves were constructed by life table method and compared with Wilcoxon test. RESULTS: Under the dosages used, there was neither mortality nor cirrhosis in AA and silica-treated groups. DDC and ANIT caused cirrhosis within 4-12 and 12-24 wk, respectively. Both showed significantly faster cirrhosis induction at high dosages without significant alteration of survival. The duration for cirrhosis induction by CCl(4) ranged from 4 to 20 wk, mainly dependent upon the dosage. However, the increase in CCl(4) dosage significantly worsened survival. Intraperitoneal CCl(4) administration resulted in better survival in comparison with gavage administration at high dosage, but not at medium and low dosages. After CBDL, all the mice developed liver cirrhosis within 4-8 wk and then died by the end of 16 wk. CONCLUSION: CBDL and administrations of ANIT, CCl(4), and DDC ensured liver cirrhosis. CBDL required the least amount of time in cirrhosis induction, but caused shortened lives of mice. It was followed by DDC and ANIT administration with favorable survival. As for CCl(4), the speed of cirrhosis induction and the mouse survival depended upon the dosages and the administration route.

Hepatic porphyria induced by the herbicide tralkoxydim in small mammals is species-specific.

Tralkoxydim is the active ingredient in a postemergent herbicide used in cereal crops. During preregistration trials, tralkoxydim was observed to cause hepatic porphyria and cholestasis in laboratory mice. Porphyria was not seen in similarly exposed rats or hamsters, but data were not collected regarding the susceptibility of any wild small mammal species to the tralkoxydim-induced porphyria. To address this data gap, we exposed small mammals to tralkoxydim, to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; a known porphyrinogenic chemical), or to sunflower oil alone. We studied small mammal species that might be exposed following agricultural applications of the herbicide, including the white-footed mouse (Peromyscus leucopus), the deer mouse (P. maniculatus), and the meadow vole (Microtus pennsylvanicus). Because of their known susceptibility to both tralkoxydim- and DDC-induced porphyria, commercially supplied Mus musculus (CD-1 Swiss mice) were exposed as positive-control animals. We also exposed offspring of wild-caught M. musculus to compare their responses to those of the commercially supplied animals. Potential hepatotoxicity was determined by assessing the accumulation of liver protoporphyrin. Of the species tested, only M. musculus was susceptible to the porphyrinogenic action of tralkoxydim, and no significant accumulation of protoporphyrin was observed in any of the other species exposed to the herbicide.

The proteasome inhibitor, PS-341, causes cytokeratin aggresome formation.

Mallory body (MB) experimental induction takes 10 weeks of drug ingestion. Therefore, it is difficult to study the dynamics and mechanisms involved in vivo. Consequently, an in vitro study was done using primary tissue culture of hepatocytes from drug-primed mice livers in which MBs had already formed. The hypothesis to be tested was that MBs are cytokeratin aggresomes, which form when hepatocytes have a defective ubiquitin-proteasome pathway by which turnover of cytokeratin proteins is prevented. To test this hypothesis, primary tissue cultures of the hepatocytes from normal and MB-forming livers were incubated with the proteasome inhibitor PS-341 and then the cytokeratin filaments and the filament connecting proteins, that is, beta-actin, and ZO1, were visualized by immunofluorescence microscopy. PS-341 caused detachment of the cytokeratins from the cell surface plasma membrane. The cytokeratin filaments retracted toward the nucleus and cytokeratin aggresomes formed. In human livers, MBs showed colocalization of cytokeratin-8 (CK-8) with ubiquitin but not with beta-actin or ZO1. Mouse hepatoma cell lines were studied using PS-341 to induce cytokeratin aggresome formation. In these cell lines, the cytokeratin filaments first retracted toward the nucleus then formed cytokeratin-ubiquitin aggresomes polarized at one side of the nucleus. At the same time, the cells became dissociated from each other, however. The results simulated MB formation. MBs differ from cytokeratin aggresomes both morphologically and in ultrastructure.

The origin and liver repopulating capacity of murine oval cells.

The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor/stem cells. To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and a method for their isolation was developed. Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver repopulation. In competitive repopulation experiments, hepatic oval cells were at least as efficient as mature hepatocytes in repopulating the liver. In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver nonparenchymal cells. This finding supports a model in which intrahepatic progenitors differentiate into hepatocytes irreversibly. To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for 8 months and oval cells were then serially transferred into Fah mutants. The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor. We conclude that hepatic oval cells do not originate in bone marrow but in the liver itself, and that they have valuable properties for therapeutic liver repopulation.

Poly(ADP-ribose) synthetase activation mediates mitochondrial injury during oxidant-induced cell death.

Reactive oxidant species are important mediators of tissue injury in shock, inflammation, and reperfusion injury. The actions of a number of these oxidants (e.g., hydroxyl radical and peroxynitrite, a reactive oxidant produced by the reaction of nitric oxide and superoxide) are mediated in part by the activation of the nuclear nick sensor enzyme, poly(ADP)-ribose synthetase (PARS), with consequent cellular energy depletion. Here we investigated whether PARS activation contributes to the mitochondrial alterations in cells exposed to oxidants. Authentic peroxynitrite (20 microM), the peroxynitrite-generating compound 3-morpholinosidnonimine, the combination of pyrogallol and S-nitroso-N-acetyl-D,L-penicillamine, as well as hydrogen peroxide induced a time- and dose-dependent decrease in mitochondrial transmembrane potential (delta psi(m)) in thymocytes, as determined by flow cytometry using the mitochondrial potential sensitive dyes DiOC6(3) and JC-1. A time- and dose-dependent increase in secondary reactive oxygen intermediate production and loss of cardiolipin, an indicator of mitochondrial membrane damage, were also observed, as measured by flow cytometry using the fluorescent dyes dihydroethidine and nonyl-acridine orange, respectively. Inhibition of PARS by 3-aminobenzamide or 5-iodo-6-amino-1,2-benzopyrone attenuated peroxynitrite-induced delta psi(m) reduction, secondary reactive oxygen intermediate generation, cardiolipin degradation, and intracellular calcium mobilization. Furthermore, thymocytes from PARS-deficient animals were protected against the peroxynitrite- and hydrogen peroxide-induced functional and ultrastructural mitochondrial alterations. In conclusion, mitochondrial perturbations during oxidant-mediated cytotoxicity are, to a significant degree, related to PARS activation rather than to direct effects of the oxidants on the mitochondria.

Effects of clonidine in a primed rat model of acute hepatic porphyria.

Acute hepatic porphyrias can be induced by several drugs and acute attacks of porphyrias are often associated with severe hypertension. Therefore it is important to know if an antihypertensive drug used has porphyrogenic potency or not. As previously demonstrated in normal rats the alpha-receptor blocker clonidine (CAS 4205-90-7) has no significant influence on the porphyrin metabolism. Pretreatment of rats with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or allyl-isopropyl-acetamide (AIA) induces hepatic delta-aminolaevulinic acid synthase (ALA-S) and increases the urinary excretion of porphyrin precursors (ALA and PBG) comparable to the latent phase of acute hepatic porphyrias in humans. Clonidine did not induce hepatic ALA-S or urinary excretion of ALA or PBG in normal as well as in DDC or AIA pretreated rats. Moreover the induction of P4501A1 (7-ethoxyresorufin-O-deethylase) by DDC was abolished by simultaneous application of clonidine. From these findings one can probably conclude that clonidine is a safe drug in human acute hepatic porphyria.

Evaluation of the porphyrinogenic risk of antineoplastics.

The use of antineoplastics is common in cancer therapy, and some of them have been associated with the development of porphyria in patients with cancer. However, knowledge of their effects on the haeme metabolic pathway is at present scarce and unclear. So, the present study evaluates the porphyrinogenic ability of nine antineoplastics (both alkylating and non-alkylating). These were tested either alone or in conjunction with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (latent porphyria model) in chick embryos and in mice. The results obtained suggest that the use of cyclophosphamide, azathioprine, 5-fluorouracil, busulphan, procarbazine and hexamethylmelamine be avoided in the treatment of porphyric patients. On the other hand, dacarbazine, chlorambucil and melphalan are non-porphyrinogenic. We also provide evidence showing that neither the presence of the mustard group in the structure of the antineoplastic nor alterations in ferrochelatase or protoporphyrinogen oxidase activities are responsible for the porphyrinogenic ability of cyclophosphamide.

Inhibition of ferrochelatase and accumulation of porphyrins in mouse hepatocyte cultures exposed to porphyrinogenic chemicals.

The ability of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine (EDDC) and griseofulvin to induce porphyria in primary cultures of mouse hepatocytes has been examined. Exposure of cultured mouse hepatocytes to DDC, EDDC or griseofulvin resulted in a marked inhibition of ferrochelatase which was sustained over the 4-day exposure period. Maximal concentrations of DDC (25 microM), EDDC (25 microM) and griseofulvin (25 microM) resulted in 14-fold, 30-fold and 9-fold increases, respectively, in total porphyrin in the culture medium. Analysis of the porphyrins accumulating indicated a predominance of protoporphyrin with all three xenobiotics. Addition of 5-aminolaevulinic acid (ALA) to mouse hepatocyte cultures (10-1000 microM) resulted in much larger increases (up to 164-fold) in porphyrin accumulation in the medium and the porphyrin accumulating was predominantly uroporphyrin. These studies have demonstrated that primary cultures of mouse hepatocytes provide a valid mechanism-based in vitro model of the hepatic porphyrias produced by the dihydropyridines and griseofulvin in mice.