ddC Exposure Research Articles

WNT7B Regulates Cholangiocyte Proliferation and Function During Murine Cholestasis.

We previously identified an up‐regulation of specific Wnt proteins in the cholangiocyte compartment during cholestatic liver injury and found that mice lacking Wnt secretion from hepatocytes and cholangiocytes showed fewer proliferating cholangiocytes and high mortality in response to a 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) diet, a murine model of primary sclerosing cholangitis. In vitro studies demonstrated that Wnt7b, one of the Wnts up‐regulated during cholestasis, induces proliferation of cholangiocytes in an autocrine manner and increases secretion of proinflammatory cytokines. We hypothesized that loss of Wnt7b may exacerbate some of the complications of cholangiopathies by decreasing the ability of bile ducts to induce repair. Wnt7b‐flox mice were bred with Krt19‐cre mice to deplete Wnt7b expression in only cholangiocytes (CC) or with albumin‐Cre mice to delete Wnt7b expression in both hepatocytes and cholangiocytes (HC + CC). These mice were placed on a DDC diet for 1 month then killed for evaluation. Contrary to our expectations, we found that mice lacking Wnt7b from CC and HC + CC compartments had improved biliary injury, decreased cellular senescence, and lesser bile acid accumulation after DDC exposure compared to controls, along with decreased expression of inflammatory cytokines. Although Wnt7b knockout (KO) resulted in fewer proliferating cholangiocytes, CC and HC + CC KO mice on a DDC diet also had more hepatocytes expressing cholangiocyte markers compared to wild‐type mice on a DDC diet, indicating that Wnt7b suppression promotes hepatocyte reprogramming. Conclusion: Wnt7b induces a proproliferative proinflammatory program in cholangiocytes, and its loss is compensated for by conversion of hepatocytes to a biliary phenotype during cholestatic injury.

The antiretroviral 2′,3′-dideoxycytidine causes mitochondrial dysfunction in proliferating and differentiated HepaRG human cell cultures

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus infection, and their use can cause mitochondrial toxicity, including mitochondrial DNA (mtDNA) depletion in several cases. The first-generation NRTIs, including 2′,3′-dideoxycytidine (ddC), were originally and are still pursued as anticancer agents. NRTI-sensitive DNA polymerases localizing to mitochondria allow for the opportunity to poison proliferating cancer cell mtDNA replication as certain cancers rely heavily on mitochondrial functions. However, mtDNA replication is independent of the cell cycle creating a significant concern that toxicants such as ddC impair mtDNA maintenance in both proliferating and nonproliferating cells. To examine this possibility, we tested the utility of the HepaRG cell line to study ddC-induced toxicity in isogenic proliferating (undifferentiated) and nonproliferating (differentiated) cells. Following ddC exposures, we measured cell viability, mtDNA copy number, and mitochondrial bioenergetics utilizing trypan blue, Southern blotting, and extracellular flux analysis, respectively. After 13 days of 1 μM ddC exposure, proliferating and differentiated HepaRG harbored mtDNA levels of 0.9% and 17.9% compared with control cells, respectively. Cells exposed to 12 μM ddC contained even less mtDNA. By day 13, differentiated cell viability was maintained but declined for proliferating cells. Proliferating HepaRG bioenergetic parameters were severely impaired by day 8, with 1 and 12 μM ddC, whereas differentiated cells displayed defects of spare and maximal respiratory capacities (day 8) and proton-leak linked respiration (day 14) with 12 μM ddC. These results indicate HepaRG is a useful model to study proliferating and differentiated cell mitochondrial toxicant exposures.

DJ-1 deficiency attenuates expansion of liver progenitor cells through modulating the inflammatory and fibrogenic niches.

Our previous study suggested that DJ-1 has a critical role in initiating an inflammatory response, but its role in the liver progenitor cell (LPC) expansion, a process highly dependent on the inflammatory niche, remains elusive. The objective of this study is to determine the role of DJ-1 in LPC expansion. The correlation of DJ-1 expression with LPC markers was examined in the liver of patients with hepatitis B or hepatitis C virus (HBV and HCV, respectively) infection, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), nonalcoholic fatty liver disease (NAFLD), cirrhosis or hepatocellular carcinoma (HCC), respectively. The role of DJ-1 in LPC expansion and the formation of LPC-associated fibrosis and inflammation was examined in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced liver injury murine model. We also determined the ability of hepatic stellate cells (HSCs) in recruiting macrophages in DJ-1 knockout (KO) mice. The expression levels of DJ-1 were upregulated in the liver of HBV, HCV, PBC and PSC patients and DDC-fed mice. Additionally, DJ-1 expression was positively correlated with LPC proliferation in patients with liver injury and mice with DDC exposure. DJ-1 has no direct effect on LPC proliferation. Reduced activation of HSCs and collagen deposition were observed in DJ-1 KO mice. Furthermore, infiltrated CD11b+Gr-1low macrophages and pro-inflammatory factors (IL-6, TNF-α) were attenuated in DJ-1 KO mice. Mechanistically, we found that HSCs isolated from DJ-1 KO mice had decreased secretion of macrophage-mobilizing chemokines, such as CCL2 and CX3CL1, resulting in impaired macrophage infiltration. DJ-1 positively correlates with LPC expansion during liver injury. DJ-1 deficiency negatively regulates LPC proliferation by impairing the formation of LPC-associated fibrosis and inflammatory niches.

Insulin-like growth factor-1 prevents dorsal root ganglion neuronal tyrosine kinase receptor expression alterations induced by dideoxycytidine in vitro.

Dideoxycytidine (zalcitabine, ddC) produces neurotoxic effects. It is particularly important to understand the toxic effects of ddC on different subpopulations of dorsal root ganglion (DRG) neurons which express distinct tyrosine kinase receptor (Trk) and to find therapeutic factors for prevention and therapy for ddC-induced peripheral sensory neuropathy. Insulin-like growth factor-1 (IGF-1) has been shown to have neurotrophic effects on DRG sensory neurons. However, little is known about the effects of ddC on distinct Trk (TrkA, TrkB, and TrkC) expression in DRG neurons and the neuroprotective effects of IGF-1 on ddC-induced neurotoxicity. Here, we have tested the extent to which the expression of TrkA, TrkB, and TrkC receptors in primary cultured DRG neurons is affected by ddC in the presence or absence of IGF-1. In this experiment, we found that exposure of 5, 25, and 50μmol/L ddC caused a dose-dependent decrease of the mRNA, protein, and the proportion of TrkA-, TrkB-, and TrkC-expressing neurons. IGF-1 (20nmol/L) could partially reverse the decrease of TrkA and TrkB, but not TrkC, expression with ddC exposure. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10μmol/L) blocked the effects of IGF-1. These results suggested that the subpopulations of DRG neurons which express distinct TrkA, TrkB, and TrkC receptors were affected by ddC exposure. IGF-1 might relieve the ddC-induced toxicity of TrkA- and TrkB-, but not TrkC-expressing DRG neurons. These data offer new clues for a better understanding of the association of ddC with distinct Trk receptor expression and provide new evidence of the potential therapeutic role of IGF-1 on ddC-induced neurotoxicity.

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.

Repopulation of β‐catenin null liver with β‐catenin positive progenitor cells and tumor formation after long‐term exposure to DDC

Exposure to DDC diet in mice results in biliary injury that induces atypical ductular proliferation, which is followed by the appearance of oval cells and the development of fibrosis. We previously reported a potential role for β‐catenin in oval cell induction and expansion. After short‐term exposure to DDC, β‐catenin knockout liver had less A6‐positive (oval cell/biliary marker) cells than wild type. To examine the role of β‐catenin in response to long‐term injury, we exposed WT and KO mice to DDC for 80 and 150 days. After long‐term exposure, we observed the emergence of cells with scant cytoplasm and oval‐shaped nuclei, but this response was more dramatic in the KO. Contrary to short‐term exposure (<4wk), KO liver harbored significantly more A6 positive cells than WT. This coincided with increased fibrosis and serum bilirubin levels in the KO. Interestingly, as early as 80 days in the KO, and more extensively at 150 days, we find clusters of hepatocyte‐like cells, which are positive for β‐catenin. We also observed tumor formation upon gross examination of KO liver at 150 days, but not in WT. Thus, we observe a β‐catenin progenitor cell population with the KO livers after DDC exposure that undergoes chronic proliferation and results in tumorigenesis.

Role for β‐catenin in repair of biliary injury after exposure to DDC

Diverse roles for the Wnt pathway in oval cell mediated liver injury repair exist. Atypical ductular proliferation (ADP) followed by the appearance of oval cells and liver fibrosis occurs in mouse liver following DDC‐induced biliary injury. We previously noted a blunted ADP in mouse liver lacking β‐catenin. To examine whether the presence of active‐β‐catenin would provide a growth or differentiation advantage in this model, liver‐specific β‐catenin over expressing mice (TG) with a mutation at ser45 were fed the DDC diet. No difference in appearance and number of A6‐positive (oval cell/biliary marker) cells was observed in the short‐term. However, we observed an increase in A6‐positive hepatocyte‐like cells in TG as early as 14 days and a dramatic difference at 150 days. We also observe an increase in PCNA positive hepatocyte‐like cells at 28 and 150 days. Coincident with this finding, we observe decreased serum bilirubin and alkaline phosphatase levels in TG mice indicating a resolution of biliary injury. It is conceivable that β‐catenin overexpression allows the liver to recover more quickly from injury. TG mice exposed to DDC for 4 weeks followed by 2 days of normal chow had less ADP and periportal inflammation compared to WT. These findings demonstrate the dexterity of β‐catenin overexpressing hepatocytes to repair biliary injury secondary to DDC exposure.

Gender Dimorphic Formation of Mouse Mallory–Denk Bodies and the Role of Xenobiotic Metabolism and Oxidative Stress

Mallory-Denk bodies (MDBs) are keratin (K)-rich cytoplasmic hepatocyte inclusions commonly associated with alcoholic steatohepatitis. Given the significant gender differences in predisposition to human alcohol-related liver injury, and the strain difference in mouse MDB formation, we hypothesized that sex affects MDB formation. MDBs were induced in male and female mice overexpressing K8, which are predisposed to MDB formation, and in nontransgenic mice by feeding the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDB presence was determined by histologic, immunofluorescence, and biochemical analyses and correlated to liver injury using serologic and pathologic markers. Cytoskeletal and metabolic liver protein analysis, in vitro metabolism studies, and measurement of oxidative stress markers and protoporphyrin-IX were performed. Male mice formed significantly more MDBs, which was attenuated modestly by estradiol. MDB formation was accompanied by increased oxidative stress. Female mice had significantly fewer MDBs and oxidative stress-related changes, but had increased ductular reaction protoporphyrin-IX accumulation, and MDB-preventive K18 induction. Evaluation of the microsomal cytochrome-P450 (CYP) enzymes revealed significant gender differences in protein expression and activity in untreated and DDC-fed mice, and showed that DDC is metabolized by CYP3A. The changes in CYPs account for the gender differences in porphyria and DDC metabolism. DDC metabolite formation and oxidative injury accumulate on chronic DDC exposure in males, despite more efficient acute metabolism in females. Gender dimorphic formation of MDBs and porphyria associate with differences in CYPs, oxidative injury, and selective keratin induction. These findings may extend to human MDBs and other neuropathy- and myopathy-related inclusions.

Increased oval cell response and fibrosis in beta‐catenin null livers after long‐term DDC exposure

An oval cell response, or ductular reaction, occurs in the mouse liver following DDC‐induced liver injury. We previously reported a potential role for β‐catenin in oval cell induction and expansion. After short term exposure (2‐4 weeks) to DDC, β‐catenin knockout liver had less A6‐positive oval cells than wild type liver. To examine the role of β‐catenin in the response to long‐term injury, we exposed WT and KO mice to DDC for 5 months. The livers from these animals were examined histologically to examine the extent of biliary injury and oval cell response. At 5 months in both groups of animals we observed the emergence of cells with scant cytoplasm and oval‐shaped nuclei, but this response was more dramatic in the KO. We utilized the oval cell marker A6 to compare the response between the two groups. Contrary to short‐term exposure (<4wk), KO liver harbored significantly more A6 positive cells than WT. This coincided with more fibrosis in the KO than observed in WT liver. We also observed nodular formation upon macroscopic examination of the KO liver. These likely indicate continued expansion and lack of differentiation of oval cells in this model. In sum, these findings indicate diverse roles for Wnt/β ‐catenin signaling during oval cell response in mice.

Potential role for beta‐catenin in oval cell differentiation during liver injury repair

Atypical ductular proliferation (ADP) occurs in the liver following DDC‐induced liver injury. We previously noted a blunted ADP in mice liver lacking β‐catenin after short‐term DDC exposure. To examine whether the presence of active‐β ‐catenin would provide a growth or differentiation advantage to atypical ductular cells in mice, liver‐specific β‐catenin over expressing mice (TG) with a stabilizing mutation at ser45 were fed the DDC diet. No significant differences in appearance and numbers of A6‐positive cells were observed, but we did observe increased numbers of A6‐positive cells with hepatocyte‐like morphology in TG mice as early as 2 weeks and a dramatic difference at 5 months. These cells may indicate an increased potential for proliferating cells to differentiate in the setting of increased stable β‐catenin. We hypothesized that such a differentiation advantage would allow the liver to recover more quickly following a change from DDC back to a normal diet. Indeed, TG mice exposed to DDC diet for 4 weeks followed by 2 days of normal chow had significantly less A6‐positive cells and had recovered close to normal liver histology compared to what is observed in WT. In sum, these findings indicate diverse roles Wnt/β ‐catenin signaling during oval cell response in mice including a role in the differentiation process during liver repair.

Chronic and cyclical neuronal loss in hippocampal slice cultures following transient inhibition of the type 1 isoform of superoxide dismutase

Increased oxidative stress contributes to chronic neurodegenerative diseases, yet the underlying mechanisms are poorly understood. Hippocampal slice cultures prepared from 20–30-day-old mice or rats were used to model chronic neuronal loss following oxidative stress. Neuronal loss was initiated by inhibition of the antioxidant enzyme, superoxide dismutase type 1 (SOD1), using the copper chelator diethyldithiocarbamate (DDC). Continuous DDC treatment of slice cultures induced delayed neuronal loss beginning at 9 days of treatment that lasted for over 4 weeks. Neuronal loss was not uniform, rather it was cyclic: peaking at days 9–13 and at days 19–21 after DDC exposure. Neuronal loss was significantly attenuated in slice cultures that overexpress SOD1, suggesting that SOD1 inhibition was responsible. Inhibitors of nitric oxide synthase also attenuated DDC-induced neuronal loss. Chronic neuronal loss, however, did not require continuous SOD1 inhibition. Application of DDC for 13 days resulted in loss of SOD1 activity. Removal of DDC restored SOD1 activity, yet the cycles of cell loss continued until no neurons remained. Astrocyte activation was observed following the second peak of neuronal loss. Media conditioned by cultures following DDC removal induced neuronal loss and microglial activation in recipient cultures. These data suggest that slice cultures released soluble neurotoxic factor(s) following DDC removal. These data also suggest that a transient reduction of SOD1 activity leads to chronic loss of hippocampal neurons. This neuronal loss may be mediated by soluble neurotoxic factor(s) and microglial activation. Cyclical neuronal loss may also underlie chronic neurodegeneration in vivo.

2-Chloro-2'-deoxyadenosine, an antileukemic drug, has an early effect on cellular mitochondrial function.

2-Chloro-2'-deoxyadenosine [CldAdo (cladribine)], a novel effective antileukemic agent, was examined for its effects on cellular mitochondrial function and DNA content after long term (< or = 7-day) incubation of cultured CCRF-CEM human leukemia cells. Dideoxycytidine (ddC), which is known to have a delayed effect on mitochondrial DNA content, was used as a positive control to monitor mitochondrial dysfunction. CldAdo at 6-16 nM was toxic to cells within 24 hr, which is in contrast to 300 nM ddC, which had no effect on cell growth for the first 4 days of treatment. Cellular lactic acid production was used to monitor concomitant perturbations in oxidative phosphorylation during drug treatment. Unlike the delayed increase in lactate observed with ddC exposure, CldAdo-treated cells exhibited a 2-2.4-fold increase in lactate levels after 2 days of exposure to 16 nM CldAdo. By days 4 and 7, however, lactate production returned to control levels. Shorter incubations with CldAdo revealed that lactate levels began to increase within 12 hr of drug exposure, paralleling cytotoxicity. We also examined mitochondrial DNA content during drug treatment by competitive polymerase chain reaction. ddC (300 nM) reduced mitochondrial DNA levels from approximately 1000 copies/untreated cell to approximately 130 copies/cell after 7 days of exposure. In contrast, cytotoxic doses of CldAdo had little or no effect on mitochondrial DNA content during the 1-week incubation. Thus, the early CldAdo-induced perturbation of mitochondrial function was not associated with a loss of mitochondrial DNA per cell. In addition, no evidence of DNA laddering, indicative of cellular apoptosis, was detected at these dosage levels and treatment times.

The Effect of Dideoxycytidine on Lymphocyte Subpopulations in Nonhuman Primates

The Effect of Dideoxycytidine on Lymphocyte Subpopulations in Nonhuman Primates. Taylor, L. D., Binienda, Z., Schmued, L., and Slikker, W., Jr. (1994). Fundam. Appl. Toxicol. 23, 434-438.In the present study, 2′,3′dideoxycytidine (ddC), which has antiretroviral activity, was given chronically to uninfected nonhuman primates to determine whether it produces adverse immunological or hematological effects. Nine healthy adult male rhesus monkeys were divided into three groups and given the following doses of ddC in a gelatin vehicle; group A, 0.06, 6.0, 3.0, and 1.5 mg/kg; group B, 0.6 mg/kg; group C, 0 mg/kg. Blood samples were collected for hematologic analysis and flow cytometric analyses of lymphocyte subpopulations. Chronic ddC exposure did not cause significant changes in the number of red blood cells, monocytes, or reticulocytes. The number of white blood cells and neutrophils increased and these changes were observed only in group A animals at the 1.5 mg/kg dose. The most significant alterations observed were decreases in the number of T helper cells (CD4) and B cells (CD20). CD4+ and CD20+ lymphocytes exhibited dose-related shifts that were reversible over time and after drug withdrawal. The results indicate that ddC has few hematologic effects but it does have profound but transient effects on the number of cells in lymphocyte subpopulations in normal primates.

The Effect of Dideoxycytidine on Lymphocyte Subpopulations in Nonhuman Primates

In the present study, 2′,3′-dideoxycytidine (ddC), which has antiretroviral activity, was given chronically to uninfected nonhuman primates to determine whether it produces adverse immunological or hematological effects. Nine healthy adult male rhesus monkeys were divided into three groups and given the following doses of ddC in a gelatin vehicle: group A, 0.06, 6.0, 3.0, and 1.5 mg/kg; group B, 0.6 mg/kg; group C, 0 mg/kg. Blood samples were collected for hematologic analysis and flow cytometric analyses of lymphocyte subpopulations. Chronic ddC exposure did not cause significant changes in the number of red blood cells, monocytes, or reticulocytes. The number of white blood cells and neutrophils increased and these changes were observed only in group A animals at the 1.5 mg/kg dose. The most significant alterations observed were decreases in the number of T helper cells (CD4) and B cells (CD20). CD4+ and CD20+ lymphocytes exhibited dose-related shifts that were reversible over time and after drug withdrawal. The results indicate that ddC has few hematologic effects but it does have profound but transient effects on the number of cells in lymphocyte subpopulations in normal primates.