Effects of acetaminophen and nonsteroidal anti-inflammatory drugs on progesterone production by porcine granulosa cells in vitro

We investigated the effects of a group of pharmaceutical agents commonly ingested by reproductive-aged women, acetaminophen and the nonsteroidal anti-inflammatory drugs (NSAID), on progesterone (P) production by cultures of highly differentiated porcine granulosa cells. These compounds were added to cultures over a dose range of 10⁻⁸ to 10⁻⁵ M and P, and cell protein was measured after 24 hours. P production was suppressed by acetaminophen, fenoprofen, and sulindac to a maximum of 81%, 76%, and 71% of control, respectively. P production was enhanced by butazolidin at all doses tested to a maximum of 140% of control. Granulosa cell protein was suppressed by butazolidin and salicylic acid to a maximum of 81% of controls. These data imply that acetaminophen and several NSAID have the potential for clinical reproductive toxicity with differing individual effects on reproductive tract tissues, suggesting further selective testing in vivo.     

Synthetic estrogens suppress granulosa cell progesterone production in vitro

We evaluated the effect of commonly used pharmaceutical estrogens and zeranol, an estrogenic growth-promoting agent used in livestock, on progesterone (P) production by cultures of highly differentiated porcine granulosa cells (GC). The compounds were added to GC cultures over a dose range of 10(-8) to 10(-5) M with P and cell protein measured after 24 h. P production was suppressed by estradiol (minimal suppressive dose: 10(-7) M; maximal suppression to 11% of control), ethinyl estradiol (10(-7) M, 15%), diethylstilbestrol (10(-5) M, 72%), clomiphene citrate (10(-6) M, 30%), nafoxidine (10(-7) M, 33%), tamoxifen (10(-6) M, 37%), and zeranol (10(-5) M, 83%). P production was not suppressed by mestranol. GC protein was suppressed by estradiol, ethinyl estradiol, nafoxidine, and zeranol. These data suggest that synthetic estrogens have the potential to suppress luteal P production by a mechanism unrelated to the usual measures of estrogenicity.     

Potential role of activated macrophages in acetaminophen hepatotoxicity. II. Mechanism of macrophage accumulation and activation

Treatment of rats with acetaminophen (1.2 g/kg) results in the accumulation of activated macrophages in the centrilobular regions of the liver. To study the mechanism by which these cells accumulate and become activated, we examined the release of chemotactic and activating factors from cultured hepatocytes treated with acetaminophen (10-100 microM). We found a dose- and time-related generation of Kupffer cell and monocyte chemotactic activity by acetaminophen-treated hepatocytes. The maximum response was observed with a 25% dilution of medium collected 24 hr following treatment of hepatocytes with acetaminophen. Using a checkerboard assay, the factor in conditioned medium was determined to induce chemotaxis as well as chemokinesis in both Kupffer cells and monocytes. The hepatocyte-derived chemotactic factor was also found to be stable to freeze-thawing but to lose activity following heat or trypsin treatment. These results, together with our findings that chemotactic activity was eluted in the void volume following Sephadex G-25 size exclusion chromatography, suggested that the chemotactic factor released by hepatocytes is a large molecular weight protein. The release of Kupffer cell activating factors by acetaminophen-treated hepatocytes was also examined. Hepatocyte-conditioned medium was found to stimulate Kupffer cell phagocytosis and superoxide anion release, two characteristics of activated macrophages. These effects were maximal with conditioned medium collected from hepatocytes 24 hr following treatment with 50-100 microM acetaminophen. Acetaminophen alone had no effect on chemotaxis, phagocytosis, or superoxide anion production by Kupffer cells or monocytes. Taken together, these results suggest that macrophage accumulation and activation in the liver following acetaminophen treatment may be mediated, at least in part, by factors released from hepatocytes.     

Effect of triptolide on progesterone production from cultured rat granulosa cells

Triptolide(CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F (TWHF), is known to have multiple pharmacological activities. However, studies have also shown that triptolide is highly disrupt to the reproductive system by disrupting normal steroid hormone signaling. In the present study, we investigated the effect of triptolide (5, 10, or 20 nM for 24 h) on progesterone production by rat granulosa cells. Triptolide inhibited both basal and human chorionic gonadotropin (HCG)- and 8-bromo-cAMP-stimulated progesterone production as revealed by RIA assay. Furthermore, the HCG-evoked increase in cellular cAMP content was also inhibited by triptolide, indicating that disruption of the cAMP/PKA signaling pathway may mediate the deleterious effects of triptolide on progesterone regulation. In addition, triptolide inhibited 25-OH-cholesterol-stimulated progesterone production, suggesting that activity of the P450 side chain cleavage (P450scc) enzyme was also be inhibited by triptolide. Western blot and quantitative real-time PCR (qRT-PCR) assays further revealed that triptolide decreased mRNA and protein expression of P450scc and the steroidogenic regulatory (StAR) protein in granulosa cells. In contrast, cell viability tests using 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) indicated that triptolide did not cause measurable cell death at doses that suppressed steroidogenesis. The reproductive toxicity of triptolide may be caused by disruption of cAMP/PKA-mediated expression of a number of progesterone synthesis enzymes or regulatory proteins, leading to reduced progesterone synthesis and reproductive dysfunction.     

Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity

Primary human hepatocytes (PHH) are the “gold standard” for in vitro toxicity tests. However, 2D PHH cultures have limitations that are due to a time-dependent dedifferentiation process visible by morphological changes closely connected to a decline of albumin production and CYP450 activity. The 3D in vitro culture corresponds to in vivo-like tissue architecture, which preserves functional characteristics of hepatocytes, and therefore can at least partially overcome the restrictions of 2D cultures. Consequently, several drug toxicities observed in vivo cannot be reproduced in 2D in vitro models, for example, the toxic effects of acetaminophen. The objective of this study was to identify molecular differences between 2D and 3D cultivation which explain the observed toxicity response. Our data demonstrated an increase in cell death after treatment with acetaminophen in 3D, but not in 2D cultures. Additionally, an acetaminophen concentration-dependent increase in the CYP2E1 expression level in 3D cultures was detected. However, during the treatment with 10 mM acetaminophen, the expression level of SOD gradually decreased in 3D cultures and was undetectable after 24 h. In line with these findings, we observed higher import/export rates in the membrane transport protein, multidrug resistance-associated protein-1, which is known to be specific for acetaminophen transport. The presented data demonstrate that PHH cultured in 3D preserve certain metabolic functions. Therefore, they have closer resemblance to the in vivo situation than PHH in 2D cultures. In consequence, 3D cultures will allow for a more accurate hepatotoxicity prediction in in vitro models in the future.     

Reduced hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase: potential role of tumor necrosis factor-alpha and interleukin-10

Macrophage-derived inflammatory mediators have been implicated in tissue injury induced by a number of hepatotoxicants. In the present studies, we used transgenic mice with a targeted disruption of the gene for inducible nitric oxide synthase (NOS II) to analyze the role of nitric oxide in inflammatory mediator production in the liver and in tissue injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis, which was evident within 3 h and reached a maximum at 18 h. This was correlated with NOS II expression and nitrotyrosine staining of the liver, which was most prominent after 6 h. Expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), matrix metalloproteinase-9, and connective tissue growth factor (CTGF) also increased in the liver following acetaminophen treatment of wild-type mice. NOS II knockout mice were found to be less sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This did not appear to be due to differences in acetaminophen-induced glutathione depletion or adduct formation. In NOS II knockout mice treated with acetaminophen, hepatic expression of TNF-alpha, as well as CTGF, was significantly increased compared to wild-type mice. In contrast, IL-10 expression was reduced. These data demonstrate that nitric oxide is important in hepatotoxicity induced by acetaminophen. Moreover, some of its effects may be mediated by altering production of pro- and antiinflammatory cytokines and proteins important in tissue repair.     

Protective effect of s-allyl cysteine and s-propyl cysteine on acetaminophen-induced hepatotoxicity in mice.

In vivo protective effects of s-allyl cysteine (SAC) and s-propyl cysteine (SPC) against acetaminophen-induced hepatotoxicity in Balb/cA mice were studied. SAC and SPC at 1g/L were added into drinking water for four weeks and followed by acetaminophen treatment. Acetaminophen treatment significantly depleted glutathione content, increased oxidation stress and elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities (P < 0.05); however, the intake of SAC or SPC significantly alleviated glutathione depletion and the elevation of ALT and AST, enhanced glutathione peroxidase activity, and lowered malondialdehyde formation (P < 0.05). Plasma levels of C-reactive protein (CRP), von Willebrand factor (vWF), IL-6, IL-10 and TNF-alpha were significantly increased by acetaminophen treatment (P < 0.05); and SAC or SPC intake significantly suppressed acetaminophen-induced elevation of CRP, vWF and the three cytokines (P < 0.05). Acetaminophen treatment also significantly increased plasminogen activator inhibitor-1 (PAI-1) activity and plasma fibrinogen level, and decreased antithrombin III (AT-III) and protein C activities (P < 0.05). SAC or SPC intake alleviated AT-III and protein C reduction (P < 0.05); but did not affect PAI-1 activity and plasma fibrinogen level (P > 0.05). These data suggest that SAC and SPC are potential multiple-protective agents against acetaminophen-induced hepatotoxicity.     

Effects of a competitive inhibitor (mevinolin) of 3-hydroxy-3-methylglutaryl coenzyme A reductase on human and bovine endothelial cells, fibroblasts and smooth muscle cells in vitro

Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, is a potent inhibitor of cholesterol synthesis. We have tested the effects of mevinolin on cell replication (3H-thymidine incorporation), prostacyclin production (6-keto-PGF1 alpha) and cell death (51Cr release) in cell cultures (human umbilical vein endothelial cells, bovine endothelial cells, human fibroblasts and bovine smooth muscle cells). Mevinolin concentrations ranging from 0.05 mumol/l (reported therapeutic concentration) to 20 mumol/l were used. In human endothelial cells the replication was reduced by 11% at a concentration of 2.0 mumol/l (P less than 0.01). In fibroblasts and smooth muscle cells the reduction was significant already at 0.1 mumol/l (10%, P less than 0.01). The prostacyclin production was reduced in endothelial cells at 1.0 mumol/l (19%, P less than 0.01) and in smooth muscle cells at 2.0 mumol/l (15%, P less than 0.05). At 20 mumol/l both cell replication and prostacyclin production was markedly reduced by about 40% in all cell types. No effects on 51Cr release or trypan blue staining was seen at any concentration. It is concluded that mevinolin has an effect on DNA synthesis and prostacyclin production on the tested cell types in vitro. These effects were, however, observed only at concentrations higher than those recommended for therapeutical use.     

Proton pump inhibitor co-therapy normalizes the increased cell turnover of the gastric mucosa both in NSAID and selective COX-2 users

Proton pump inhibitor (PPI) co-therapy is considered the best strategy in preventing gastrointestinal complications during non-steroidal anti-inflammatory drug (NSAID) treatment, but there is limited information available on its effect on gastric mucosal cell kinetics. To evaluate the effect of PPI co-therapy on gastric mucosa we investigated epithelial cell proliferation, apoptosis, epithelial growth factor receptor (EGFR) and p53 expression in patients on chronic non-selective NSAID or cyclooxygenase-2 selective inhibitor (COX-2) treatment. Gastric biopsies of the antrum were taken from 10-10 patients on chronic NSAID and COX-2, therapy prior and after 6 months PPI co-therapy, and 10 controls without any treatment. Cell proliferation, apoptosis, EGFR and p53 expression were measured by immunohistochemistry. At least 600 glandular epithel cells were encountered and results were expressed as % of total cells counted. We found increased cell proliferation in patients on chronic COX-2 but not on NSAID therapy. Patients on either NSAID or COX-2 therapy had an increased p53 and decreased EGFR expression. PPI therapy reversed not only the increased cell proliferation and p53 expression, but also the suppressed EGFR expression when administered as co-therapy. The fewer gastrointestinal side effects observed during chronic COX-2 therapy may partially be the result of the higher cell proliferation. This effect is not mediated by the EGFR pathway. PPI co-therapy normalizes the disturbed cell kinetics irrespective of NSAID treatment used.     

Effects of sulfur-amino acid-deficient diets on acetaminophen metabolism and hepatotoxicity in rats

Cysteine is required for the synthesis of cosubstrates for two pathways of acetaminophen metabolism: 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for sulfation and glutathione (GSH) for detoxification of the reactive metabolite (N-acetyl-p-benzoquinoneimine, NAPQI). Dietary deficiency of cysteine may reduce hepatic production of PAPS and GSH and thereby reduce metabolism of the drug (by sulfation and detoxification of NAPQI) and hence lead to potentiation of acetaminophen liver injury. Conversely, limitation of sulfur-containing amino acids could result in depression of protein synthesis and hepatic cytochrome P450 levels, and hence in decreased reactive metabolite formation and decreased liver injury. To determine whether the potentiating effects exceed the protective effects, rats were fed isocaloric AIN-76 liquid diets containing various levels of methionine as the sole source of sulfur in the diet for 3 weeks prior to administration of acetaminophen. Sulfur deficiency was assessed by measuring urinary inorganic sulfate levels. Sulfur-deficient diets retarded growth but did not affect nitrogen balance. Sulfur-deficient animals had lower basal levels of hepatic GSH. Pharmacokinetic studies revealed that at low doses of acetaminophen (20 mg/kg), animals fed sulfur-deficient diets metabolized the drug more slowly due to a markedly reduced sulfation capacity, whereas at the high dose of acetaminophen (400 mg/kg), rats that were fed sulfur-deficient diets had a higher clearance of the drug than rats that were fed the complete diet. The increase in clearance was due largely to an enhanced glucuronidation capacity and an enhanced P450-dependent oxidation as indicated by mercapturate formation. Histologic studies revealed that rats fed sulfur-deficient diets showed increases in both incidence and severity of acetaminophen hepatic necrosis. Thus, the potentiating effects exceeded the protective effects. These observations raise the possibility that nutritional inadequacy of sulfur-containing amino acids which could occur during protein malnutrition may similarly enhance susceptibility to acetaminophen liver injury in humans.     

Acetaminophen analgesia in children: placebo effect and pain resolution after tonsillectomy

BACKGROUND: Pharmacodynamic models of acetaminophen analgesia in children have not explored the efficacy of single oral doses greater than 40 mg/kg. METHODS: Children aged 9.0 +/- 3.0 years (+/- SD) and weight 37.9+/- 16.6 kg undergoing outpatient tonsillectomy were randomised to receive acetaminophen elixir 40 mg/kg (n = 12). high dose acetaminophen elixir 100 mg/kg (n =20) or placebo (n=30) 0.5 -1 h preoperatively. No other analgesics were given. Individual acetaminophen serum concentrations and pain scores [visual analogue scale (VAS) 0-10] were measured over a 4-8 h postoperative period. These data were pooled with data from a previous study investigating acetaminophen pharmacodynamics (n = 120) and analysed using a non-linear mixed effect model. Placebo effects and drug effects were modelled using effect-site concentration models. RESULTS: A one-compartment model with first-order input, lag time and first-order elimination was used to describe the population pharmacokinetics of acetaminophen. Pharmacokinetic parameter estimates were similar to those previously described. Pharmacodynamic population parameter estimates [population variability coefficient of variation (CV)] for a maximum analgesic effect (Emax) model, in which the greatest possible pain relief (VAS 0-10) equates to an Emax of 10, were Emax 5.17 (64%) and 50% effective concentration 9.98 mg/l (107%). The equilibration half-life (t(eq)) of the analgesic effect compartment was 53 min (217%). A placebo drug model for the effects of placebo response had a t(eq) of 1.96 h (40%), an elimination half-life of 2.06 h (50%) and a potency of 1.54 pain relief units (24%). CONCLUSIONS: High dose acetaminophen (100 mg/kg) was no more effective than 40 mg/kg and was associated with increased nausea and vomiting. A target effect compartment concentration of 10 mg/l is expected to produce a pain reduction of 2.6 units. The placebo model accounted for a maximum pain reduction of 5.6 units at 3 h. The combination of placebo effect and preoperative acetaminophen 40 mg/kg results in pain scores below 4 units for 5 h postoperatively.     

The covalent binding of acetaminophen to cellular nucleic acids as the result of the respiratory burst of neutrophils derived from the HL-60 cell line.

After being induced to differentiate into a neutrophilic type, cultures of the leukemic cell line HL-60 were able to cause the bioactivation and nucleic acid binding of acetaminophen upon stimulation of the respiratory burst. This phenomenon was found to simulate the same process as that previously shown with normal human granulocytes. Binding to both DNA and RNA of the cells was determined quantitatively by use of 14C-labeled acetaminophen congeners. Protein binding occurred to about the same extent as did RNA binding. Simultaneous labeling experiments with [ring-14C]- and [14C = O]acetaminophen further showed that the acetaminophen molecule was bound to DNA in an intact manner, while binding to RNA showed about a 50% excess binding of the acetaminophen ring relative to the carbonyl group. Experiments with certain inhibitors showed that catalase and azide ion strongly inhibited DNA binding, while superoxide dismutase had a slight stimulatory effect on binding. These results suggest a significant role for myeloperoxidase in the bioactivation process, which contrasts with the proposed bioactivation mechanism of certain arylamine compounds. A mechanism was proposed for acetaminophen binding to nucleic acids that requires the 1 e- oxidation of this substrate to its phenoxyl radical, although the production of the N-acetyl-p-benzoquinoneimine metabolite, which has been proposed to account for the extensive protein binding known to occur for acetaminophen, might also contribute to such binding. The potential genotoxicity of acetaminophen was considered in view of what might be a unique pathway which can metabolize this chemical to a nucleic acid-binding species.     

Endotoxin pretreatment protects against the hepatotoxicity of acetaminophen and carbon tetrachloride: role of cytochrome P450 suppression

Bacterial endotoxin (lipopolysaccharide, LPS) is known to potentiate the toxicity of many hepatotoxicants. However, exposure to a sublethal dose of LPS renders animals tolerant to a lethal dose of LPS, and protects against the toxicity of some chemicals. This study was designed to examine the effects of LPS pretreatment on acetaminophen- and carbon tetrachloride (CCl(4))-induced liver injury in LPS-sensitive C3H/OuJ and LPS-resistant C3H/HeJ mice. Pretreatment of male C3H/OuJ mice with a single injection of LPS (0. 1 mg/kg, ip, for 24 h) protected against the hepatotoxic effects of acetaminophen (400 mg/kg) and carbon tetrachloride (CCl(4), 30 mg/kg), as indicated by serum alanine aminotransferase activity. In contrast, pretreatment of C3H/HeJ mice with 0.1 or 10 mg/kg LPS afforded no protection against the hepatotoxic effects of acetaminophen and CCl(4). In an attempt to determine the mechanism of LPS-induced protection against acetaminophen- and CCl(4)-induced hepatotoxicity in C3H/OuJ mice, liver cytochrome P450 was determined 24 h after LPS injection. LPS treatment caused a 26% decrease in total P450 content in C3H/OuJ but not in C3H/HeJ mice. CYP3A-catalized testosterone 6 beta-, 2 beta-, and 15 beta-hydroxylation was decreased 40% by LPS only in C3H/OuJ mice. To determine whether the differences to LPS-response in the two stains of mice is mediated by a strain-related difference in the release of cytokines, mice were pretreated with interleukin-1 (IL-1 alpha, 5 x 10(5) U/mouse), and the hepatoprotection and hepatic P450 enzymes were examined. IL-1 alpha pretreatment equally protected against the hepatotoxicity of acetaminophen and CCl(4) in both strains, and suppressed the total microsomal P450 and P450 enzyme-catalyzed testosterone hydroxylation to a similar extent. In conclusion, LPS pretreatment suppressed hepatic cytochrome P450 enzymes and protected against the hepatotoxicity of acetaminophen and CCl(4) in LPS-sensitive C3H/OuJ mice, but not in LPS-refractory C3H/HeJ mice. This protective effect of LPS appears to be mediated through the release of cytokines such as IL-1 alpha, which in turn suppresses the cytochrome P450 responsible for the activation of acetaminophen and CCl(4) to reactive metabolites.     

Protective effect of fucoidan against acetaminophen-induced liver injury

Fucoidan, a sulfated polysaccharide extracted from various brown seaweeds, possesses a wide range of pharmacological properties. In this study, we investigated the protective effect of fucoidan on acetaminophen-induced acute liver injury in rats. Liver injury was induced by administration of acetaminophen (800 mg/kg, i.p.) and fucoidan was administered (100 mg kg, p.o.) 2 h before and after acetaminophen administration. After 24 h, co-treatment of fucoidan ameliorated liver damage and cell death induced by acetaminophen. Acetaminophen induced the overexpression of CYP2E1, one of the metabolizing enzymes of acetaminophen, but cotreatment with fucoidan suppressed its increased expression of CYP2E1. Fucoidan also reduced the hepatic apoptosis induced by acetaminophen exposure as shown in the protein expression of Bax, Bcl-2, and cleaved caspase-3. The anti-oxidative effect of fucoidan was observed from the increase of the production and expression of glutathione, superoxide dismutase, and glutathione peroxidase, both of which were decreased by acetaminophen. Also, fucoidan decreased the expression of inflammatory mediators, including tumor necrosis factoralpha, interleukin 1 beta, and inducible nitric oxide synthase. Taken together, the data demonstrate the hepato-protective effects of fucoidan against acetaminophen-induced liver injury via anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.     

Acetaminophen exhibits weak antiestrogenic activity in human endometrial adenocarcinoma (Ishikawa) cells.

The purpose of this study was to test the hypothesis that acetaminophen would alter an estrogen-regulated process in human cells that express endogenous estrogen receptor alpha and beta (ERalpha and ERbeta). Specifically, the extent to which acetaminophen altered the expression of estrogen-inducible alkaline phosphatase in endometrial adenocarcinoma (Ishikawa) cells and directly interacted with ERbeta and ERalpha was determined. Ishikawa cells were exposed to estradiol and/or to a range of concentrations of acetaminophen for four days, and alkaline phosphatase activity was measured spectrophotometrically. Acetaminophen inhibited both basal and estradiol-induced alkaline phosphatase activity in Ishikawa cells in a concentration-dependent manner. The reduction of Ishikawa cell alkaline phosphatase was not due to direct inhibition of enzyme activity by acetaminophen. Toxic effects of acetaminophen on Ishikawa cells were determined by measuring loss of cellular lactate dehydrogenase to culture medium. High concentrations of acetaminophen (>/=0.5 mM) induced lactate dehydrogenase release from cells and reduced the amount of cellular protein in culture dishes, indicating some acetaminophen-induced reduction of alkaline phosphatase activity might be attributed to toxic effects. However, lower concentrations of acetaminophen significantly reduced alkaline phosphatase activity in the absence of detectable toxicity. Acetaminophen also augmented 4-hydroxy-tamoxifen reduction of alkaline phosphatase activity. Competition binding assays with human ERalpha and ERbeta demonstrated 10(6)-fold molar excess acetaminophen did not directly interact significantly with the ligand-binding domain of either receptor. These studies indicate acetaminophen exerts weak antiestrogenic activity in Ishikawa cells without directly binding ERalpha or ERbeta.     

Acetaminophen alters estrogenic responses in vitro: inhibition of estrogen-dependent vitellogenin production in trout liver cells.

The purpose of this study was to determine if acetaminophen altered estrogen-dependent vitellogenin production in isolated trout liver cells. Estrogen-induced vitellogenesis was studied in liver cells isolated from male trout and cultured in defined medium; vitellogenin secreted into culture medium was quantitated using immunological procedures. Vitellogenin production was absolutely dependent on the addition of estradiol (10(-6) M) to liver cells from male trout. Acetaminophen produced a dose-dependent inhibition of vitellogenin production; approximately 50% inhibition was achieved with 0.05 mM acetaminophen, while 0.3 mM acetaminophen inhibited secreted vitellogenin to undetectable levels. In contrast, these concentrations of acetaminophen (< or = 1 mM) did not significantly alter the production of secreted albumin, determined immunologically, or cause detectable toxicity. Higher doses of acetaminophen were toxic, but did not induce DNA fragmentation in the trout liver cells. Acetaminophen reduction of estradiol-induced vitellogenin production was accompanied by a dose-dependent decrease in vitellogenin mRNA, indicating acetaminophen inhibited a step prior to, or during, formation of vitellogenin mRNA. Estrogen receptor-binding assays demonstrated that acetaminophen did not reduce binding of [3H]-estradiol to trout liver estrogen receptor. In addition, catabolism of estradiol to water-soluble metabolites was not significantly altered by acetaminophen. These studies indicate that non-toxic concentrations of acetaminophen specifically inhibit estrogen-dependent vitellogenin synthesis and suggest that this commonly used drug may alter estrogen-regulated processes.     

In vitro effect of amantadine and interferon alpha-2a on hepatitis C virus markers in cultured peripheral blood mononuclear cells from hepatitis C virus-infected patients.

The effects of amantadine (1-5 microM) and interferon alpha (IFNalpha)-2a alone (1000 IU/ml) and combined, have been studied in cultured peripheral blood mononuclear cells (PBMC) from 15 chronic hepatitis C patients and ten healthy donors. Amantadine itself did not affect cell viability and had minor effects on the response to mitogens by PBMC. Four patients (27%), but no donors, had hepatitis C virus (HCV) core and NS3-specific proliferative responses. Amantadine suppressed these responses in all cases and its antiproliferative effect was greater than that of IFNalpha (Mann-Whitney's U-test: P < 0.05 in both cases). All PBMC cultures from patients, but none from donors, were HCV RNA positive. Amantadine alone or combined with IFNalpha dose-dependently reduced HCV RNA content in individual PBMC (Wilcoxon's signed rank test: 1 microM, P < 0.05; 2 microM, P < 0.02; and 5 microM, P = 0.16) with respect to untreated cultures. In addition, 7, 13 and 20% of PBMC cultures became HCV RNA negative with 2 microM amantadine alone, IFNalpha alone and their combination, respectively. Finally, in contrast to IFNalpha, amantadine did not modify expression of 2',5'-oligoadenylate synthetase activity or the spontaneous or mitogen-stimulated IFNgamma and interleukin 10 production. In conclusion, these effects in PBMC from HCV patients suggest that the amantadine/IFNalpha combination might be considered a therapeutic option for treating chronic hepatitis C patients.