Evaluation of potassium iodide (KI) and ammonium perchlorate (NH4ClO4) to ameliorate 131I- exposure in the rat.

Nuclear reactor accidents and the threat of nuclear terrorism have heightened the concern for adverse health risks associated with radiation poisoning. Potassium iodide (KI) is the only pharmaceutical intervention that is currently approved by the Food and Drug Administration for treating (131)I(-) exposure, a common radioactive fission product. Though effective, KI administration needs to occur prior to or as soon as possible (within a few hours) after radioactive exposure to maximize the radioprotective benefits of KI. During the Chernobyl nuclear reactor accident, KI was not administered soon enough after radiation poisoning occurred to thousands of people. The delay in administration of KI resulted in an increased incidence of childhood thyroid cancer. Perchlorate (ClO(4)(-)) was suggested as another pharmaceutical radioprotectant for 131I- poisoning because of its ability to block thyroidal uptake of iodide and discharge free iodide from the thyroid gland. The objective of this study was to compare the ability of KI and ammonium perchlorate to reduce thyroid gland exposure to radioactive iodide (131I-). Rats were dosed with 131I- tracer and 0.5 and 3 h later dosed orally with 30 mg/kg of either ammonium perchlorate or KI. Compared to controls, both anion treatments reduced thyroid gland exposure to 131I- equally, with a reduction ranging from 65 to 77%. Ammonium perchlorate was more effective than stable iodide for whole-body radioprotectant effectiveness. KI-treated animals excreted only 30% of the (131)I(-) in urine after 15 h, compared to 47% in ammonium perchlorate-treated rats. Taken together, data suggest that KI and ammonium perchlorate are both able to reduce thyroid gland exposure to 131I- up to 3 h after exposure to 131I-. Ammonium perchlorate may offer an advantage over KI because of its ability to clear 131I- from the body.

Adenosine A(2A) and A(2B) receptor activation of erythropoietin production.

We have examined the effects of adenosine receptors and protein kinases A and C in the regulation of erythropoietin (Epo) production using hepatocellular carcinoma (Hep3B) cells in culture and in vivo in normal mice under normoxic and hypoxic conditions. CGS-21680, a selective adenosine A(2A) agonist, significantly increased levels of Epo in normoxic Hep3B cell cultures and in serum of normal mice under both normoxic and hypoxic conditions. CGS-21680 also produced a significant increase in Epo mRNA levels in Hep3B cell cultures. SCH-58261, a selective adenosine A(2A) receptor antagonist, significantly inhibited the increase in medium levels of Epo in Hep3B cell cultures exposed to hypoxia (1% O(2)). Enprofylline, a selective adenosine A(2B) receptor antagonist, significantly inhibited the increase in plasma levels of Epo in normal mice exposed to hypoxia. Chelerythrine chloride, an antagonist of protein kinase C activation, significantly inhibited hypoxia-induced increases in serum levels of Epo in normal mice. A model is presented for adenosine in hypoxic regulation of Epo production that involves kinases A and C and phospholipase A(2) pathways.

Metabolism of trichloroethylene.

A major focus in the study of metabolism and disposition of trichloroethylene (TCE) is to identify metabolites that can be used reliably to assess flux through the various pathways of TCE metabolism and to identify those metabolites that are causally associated with toxic responses. Another important issue involves delineation of sex- and species-dependent differences in biotransformation pathways. Defining these differences can play an important role in the utility of laboratory animal data for understanding the pharmacokinetics and pharmacodynamics of TCE in humans. Sex-, species-, and strain-dependent differences in absorption and distribution of TCE may play some role in explaining differences in metabolism and susceptibility to toxicity from TCE exposure. The majority of differences in susceptibility, however, are likely due to sex-, species-, and strain-dependent differences in activities of the various enzymes that can metabolize TCE and its subsequent metabolites. An additional factor that plays a role in human health risk assessment for TCE is the high degree of variability in the activity of certain enzymes. TCE undergoes metabolism by two major pathways, cytochrome P450 (P450)-dependent oxidation and conjugation with glutathione (GSH). Key P450-derived metabolites of TCE that have been associated with specific target organs, such as the liver and lungs, include chloral hydrate, trichloroacetate, and dichloroacetate. Metabolites derived from the GSH conjugate of TCE, in contrast, have been associated with the kidney as a target organ. Specifically, metabolism of the cysteine conjugate of TCE by the cysteine conjugate ss-lyase generates a reactive metabolite that is nephrotoxic and may be nephrocarcinogenic. Although the P450 pathway is a higher activity and higher affinity pathway than the GSH conjugation pathway, one should not automatically conclude that the latter pathway is only important at very high doses. A synthesis of this information is then presented to assess how experimental data, from either animals or from (italic)in vitro (/italic)studies, can be extrapolated to humans for risk assessment. (italic)Key words(/italic): conjugate beta-lyase, cysteine glutathione, cytochrome P450, glutathione (italic)S(/italic)-transferases, metabolism, sex dependence, species dependence, tissue dependence, trichloroethylene.

Physiologically based pharmacokinetic models for trichloroethylene and its oxidative metabolites.

Trichloroethylene (TCE) pharmacokinetics have been studied in experimental animals and humans for over 30 years. Compartmental and physiologically based pharmacokinetic (PBPK) models have been developed for the uptake, distribution, and metabolism of TCE and the production, distribution, metabolism, and elimination of P450-mediated metabolites of TCE. TCE is readily taken up into systemic circulation by oral and inhalation routes of exposure and is rapidly metabolized by the hepatic P450 system and to a much lesser degree, by direct conjugation with glutathione. Recent PBPK models for TCE and its metabolites have focused on the major metabolic pathway for metabolism of TCE (P450-mediated metabolic pathway). This article briefly reviews selected published compartmental and PBPK models for TCE. Trichloroacetic acid (TCA) is considered a principle metabolite responsible for TCE-induced liver cancer in mice. Liver cancer in mice was considered a critical effect by the U.S. Environmental Protection Agency for deriving the current maximum contaminant level for TCE in water. In the literature both whole blood and plasma measurements of TCA are reported in mice and humans. To reduce confusion about disparately measured and model-predicted levels of TCA in plasma and whole blood, model-predicted outcomes are compared for first-generation (plasma) and second-generation (whole blood) PBPK models published by Fisher and colleagues. Qualitatively, animals and humans metabolize TCE in a similar fashion, producing the same metabolites. Quantitatively, PBPK models for TCE and its metabolites are important tools for providing dosimetry comparisons between experimental animals and humans. TCE PBPK models can be used today to aid in crafting scientifically sound public health decisions for TCE.

Avoiding transfusion in head and neck surgery: feasibility study of erythropoietin.

OBJECTIVE: To determine the feasibility of perioperative erythropoietin to avoid blood transfusion in head and neck cancer surgery. STUDY DESIGN: Retrospective chart review. METHODS: Ninety-nine patients undergoing surgical resection of head and neck tumors at our institution were assessed for demographic data, nutritional parameters, tumor/surgical information, hematological/transfusion data, and contraindications to erythropoietin. Each transfusion was classified as to its appropriateness, and the potential benefit of erythropoietin was assessed in each patient. A cost analysis was also performed. RESULTS: Most transfused patients (63%) received too many units. A subgroup at high risk of transfusion was identified who would benefit most from perioperative erythropoietin. Assuming that perioperative erythropoietin therapy is equivalent to the transfusion of 4 units, we estimate that the majority (741%) of transfused patients would not have required a transfusion if more stringent transfusion criteria were followed and those at high risk were given perioperative erythropoietin. Although the cost for transfusing 4 units is equivalent to that of a perioperative course of erythropoietin, the overall direct cost of erythropoietin treatment would actually have been more expensive. CONCLUSIONS: Perioperative erythropoietin therapy may be appropriate for a subgroup of head and neck cancer patients, but a prospective randomized controlled study in such a subgroup is needed to better define those most likely to benefit from it and to assess actual cost/benefit ratios.

Protein kinase C alpha protein expression is necessary for sustained erythropoietin production in human hepatocellular carcinoma (Hep3B) cells exposed to hypoxia.

Although protein kinase C (PKC) has been implicated as an effector of erythropoietin (EPO) production, its exact role is still uncertain. Hep3B human hepatocellular carcinoma cells were used for this study and were depleted of PKC in three different ways: long-term treatment with phorbol 12-myristate 13-acetate (PMA), selective inhibition with calphostin C, and treatment with PKCalpha antisense oligonucleotides. When EPO-producing Hep3B cells were incubated in 1% O2 (hypoxia) for 24 h, PMA treatment resulted in significant decreases in medium levels of EPO in Hep3B cell cultures at concentrations higher than 10 nM. The specific PKC inhibitor, calphostin C, significantly inhibited medium levels of EPO and EPO mRNA levels in Hep3B cells exposed to 1% O2. Western blot analysis revealed that Hep3B cells express the classical PKCalpha and gamma isoforms, as well as novel PKCepsilon and delta and the atypical zeta isoform. Preincubation with PMA for 6 h specifically down-regulated PKCalpha protein expression. Phosphorothioate modified antisense oligonucleotides specific for PKCalpha also decreased EPO production in Hep3B cells exposed to hypoxia for 20 h when compared to PKCalpha sense treatment. The translocation of PKCalpha from the soluble to particulate fractions was increased in Hep3B cells incubated under hypoxia compared with normoxia (21% O2) controls. These results suggest that the PKCalpha isoform plays an important role in sustaining hypoxia-regulated EPO production.

Identification of S-(1,2-dichlorovinyl)glutathione in the blood of human volunteers exposed to trichloroethylene.

Healthy male and female human volunteers were exposed to 50 ppm or 100 ppm trichloroethylene (Tri) by inhalation for 4 h. Blood and urine samples were taken at various times before, during, and after the exposure period for analysis of glutathione (GSH), related thiols and disulfides, and GSH-derived metabolites of Tri. The GSH conjugate of Tri, S-(1,2-dichlorovinyl)glutathione (DCVG), was found in the blood of all subjects from 30 min after the start of the 4-h exposure to Tri to 1 to 8 h after the end of the exposure period, depending on the dose of Tri and the sex of the subject. Male subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 2 h after the start of the exposure of 46.1 +/- 14.2 nmol/ml (n = 8), whereas female subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 4 h after the start of the exposure of only 13.4 /- 6.6 nmol/ml (n = 8). Pharmacokinetic analysis of blood DCVG concentrations showed that the area under the curve value was 3.4-fold greater in males than in females, while the t1/2 values for systemic clearance of DCVG were similar in the two sexes. Analysis of the distribution of individual values indicated a possible sorting, irrespective of gender, into a high- and a low-activity population, which suggests the possibility of a polymorphism. The mercapturates N-acetyl-1,2-DCVC and N-acetyl-2,2-DCVC were only observed in the urine of 1 male subject exposed to 100 ppm Tri. Higher contents of glutamate were generally found in the blood of females, but no marked differences between sexes were observed in contents of cyst(e)ine or GSH or in GSH redox status in the blood. Urinary GSH output exhibited a diurnal variation with no apparent sex- or Tri exposure-dependent differences. These results provide direct, in vivo evidence of GSH conjugation of Tri in humans exposed to Tri and demonstrate markedly higher amounts of DCVG formation in males, suggesting that their potential risk to Tri-induced renal toxicity may be greater than that of females.

Activation of protein kinase C in human hepatocellular carcinoma (HEP3B) cells increases erythropoietin production.

Some investigators have reported previously that phorbol esters inhibit in vitro erythropoietin production stimulated by hypoxia; whereas others have reported that phorbol esters enhanced Epo production during exposure to hypoxia. We have demonstrated in the present experiments that hypoxia significantly increased diacylglycerol levels in cultured human hepatocellular carcinoma (Hep3B) cells. 1-oleoyl-2-acetyl-ras-glycerol (OAG) and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), two well-known protein kinase C activators, significantly increased medium levels of erythropoietin as well as erythropoietin messenger RNA levels in normoxic Hep3B cells. A potent protein kinase C inhibitor, chelerythrine chloride, significantly decreased hypoxia-induced increases in medium levels of erythropoietin as well as erythropoietin messenger RNA levels in Hep3B cells. A cis-unsaturated free fatty acid, oleic acid, significantly enhanced OAG-induced medium levels of erythropoietin in normoxic Hep3B cells, whereas a phospholipase A2 inhibitor, mepacrine, significantly decreased hypoxia-induced erythropoietin production in Hep3B cells. These results provide strong support for a positive role for protein kinase C in the hypoxic regulation of erythropoietin production.

Physiologically-based pharmacokinetic model for trichloroethylene considering enterohepatic recirculation of major metabolites.

Trichloroacetic acid (TCA) is a major metabolite of trichloroethylene (TRI) thought to contribute to its hepatocarcinogenic effects in mice. Recent studies have shown that peak blood concentrations of TCA in rats do not occur until approximately 12 hours following an oral dose of TRI. However, blood concentrations of TRI reach a maximum within an hour and are nondetectable after 2 hours. The results of a study which examined the enterohepatic recirculation (EHC) of the principle TRI metabolites was used to develop a physiologically-based pharmacokinetic model for TRI, which includes enterohepatic recirculation of its metabolites. The model quantitatively predicts the uptake, distribution and elimination of TRI, trichloroethanol, trichloroethanol-glucuronide, and TCA and includes production of metabolites through the enterohepatic recirculation pathway. Physiologic parameters used in the model were obtained from the literature. Parameters for TRI metabolism were taken from Fisher et al. Other kinetic parameters were found in the literature or estimated from experimental data. The model was calibrated to data from experiments of an earlier study where TRI was orally administered. Verification of the model was conducted using data on the enterohepatic recirculation of TCEOH and TCA, chloral hydrate data (infusion doses) from Merdink, and TRI data from Templin and Larson and Bull.

A pharmacokinetic study of occupational and environmental benzene exposure with regard to gender.

Using physiologically-based pharmacokinetic (PBPK) modeling, occupational, personal, and environmental benzene exposure scenarios are simulated for adult men and women. This research identifies differences in internal exposure due to physiological and biochemical gender differences. Physiological and chemical-specific model parameters were obtained from other studies reported in the literature and medical texts for the subjects of interest. Women were found to have a higher blood/air partition coefficient and maximum velocity of metabolism for benzene than men (the two most sensitive parameters affecting gender-specific differences). Additionally, women generally have a higher body fat percentage than men. These factors influence the internal exposure incurred by the subjects and should be considered when conducting a risk assessment. Results demonstrated that physicochemical gender differences result in women metabolizing 23-26% more benzene than men when subject to the same exposure scenario even though benzene blood concentration levels are generally higher in men. These results suggest that women may be at significantly higher risk for certain effects of benzene exposure. Thus, exposure standards based on data from male subjects may not be protective for the female population.

A quest for erythropoietin over nine decades.

The major research accomplishments of the author are described from the time of his PhD thesis work on the mechanism of cobalt polycythemia to the present day. His early work on the quest for the cell that produces erythropoietin (Epo) to his current work on oxygen sensing and signal transduction pathways involved in erythropoietin gene expression are reported. He describes his main research interest in the mechanism of cobalt polycythemia between 1954 and 1962 and his research on how hormones such as the glucocorticoids function in the regulation of erythropoiesis (1956-1962). His major findings during this period were the discovery that hydrocortisone and corticosterone stimulated erythropoiesis (1958) and that cobalt increased erythropoietin production in the isolated perfused dog kidney (1961). He describes how he was led astray in some of his early studies on the cells in the kidney that produce erythropoietin, because of the less-developed technology available to him at that time; and how in situ hybridization and other molecular biology techniques enabled him to confirm some of the earlier work in mice by other investigators that interstitial cells in the kidney were the site of production of erythropoietin in the primate. His work in the controversial area of the mechanism of the anemia of end-stage renal disease is described in detail, as it pertains to Epo deficiency and suppressed erythroid progenitor cell response to Epo. He also discusses his recent work on signal transduction pathways (hypoxia, nitric oxide, adenosine, and C kinase) in oxygen sensing and Epo gene expression.

Erythropoietin: physiologic and pharmacologic aspects.

The purpose of this review is to give an update of the recent progress in research on erythropoietin (Epo), the hormone that regulates red blood cell production. Epo is a glycoprotein with a molecular mass of approx 30 kDa, which circulates in plasma of the human with 165 amino acids with three N-linked and one O-linked acidic oligosaccharide side chains in the molecule. Both the alpha (39% CHO) and beta (24% CHO) forms are available for clinical use, and there does not appear to be any difference in the pharmacokinetics of these two forms of Epo. Radioimmunoassays and enzyme-linked immunoabsorbant (ELISA) assays are available in a kit form. Serum levels of Epo in normal human subjects range between 1 and 27 mmu/ml or approx 5 pmol/l. It seems clear that the cells in the adult mammalian kidney which produce Epo are the interstitial cells in the peritubular capillary bed and the perivenous hepatocytes in the liver. Expression of the human Epo gene sequences that direct expression in the kidney are located 6-14 kilobases 5' to the gene; whereas the sequences that control hepatocyte-specific expression are located within 0.7 KS to the 3'-flanking region and 0.5 KS to the 5'-flanking region. The signal transduction pathways postulated to be involved in the expression of Epo are: kinases A, G and C; both a constitutive factor and a second hypoxia-inducible factor-1 (HIF-1) located in the 5' end of an hypoxia inducible enhancer region of the Epo gene; and reactive oxygen species. The primary target cell in the bone marrow acted on by Epo is the colony-forming unit erythroid (CFU-E) which has the highest number of Epo receptors. It has been postulated that Epo decreases the rate which Epo-dependent progenitor cells undergo programed cell death (apoptosis). There are two major signal transduction pathways activated by the Epo receptor: the JAK2-STAT5 pathway and the ras pathway. Both pathways involve tyrosine phosphorylation. The approved clinical uses of Epo are the anemias associated with end-stage renal disease, cancer chemotherapeutic agents, and patients with HIV infection receiving AZT. Other anemias reported to respond to Epo therapy are anemia of prematurity, rheumatoid arthritis, and myelodysplasia. Other uses of Epo under investigation are in perioperative surgery and preoperative autologous blood donation.

Carrier effects of dosing the H4IIE cells with 3,3',4,4'-tetrachlorobiphenyl (PCB77) in dimethyl sulfoxide or isooctane.

A rat hepatoma cell line, H4IIE, serves as a bioassay tool to assess the potential toxicity of dioxin-like chemicals, including polychlorinated biphenyls (PCB) in environmental samples. PCB exposure to these cells induces cytochrome (CYP) P4501A1 activity in a dose-dependent fashion, thus allowing assessment of mixtures. The objective of this study was to determine the effect of different carriers, dimethyl sulfoxide (DMSO) and isooctane on the concentrations of PCBs in the H4IIE cells and induction of CYP1A1 activity as measured by ethoxyresorufin O-deethylase (EROD) activity. H4IIE cells were dosed with three micrograms of UL-14C-PCB77/plate dissolved in DMSO or isooctane, and were harvested at sequential time periods for 4 days. PCB77 concentration and EROD activity were measured in the cells. EROD activity was greater when using DMSO as compared to isooctane, while there was no difference in the distribution of PCB77-derived radioactivities within the cell culture system based upon the carrier solvent used to deliver PCB77.

Inducible nitric oxide synthase expression and erythropoietin production in human hepatocellular carcinoma cells.

We have previously reported an interaction of nitric oxide (NO) and cyclic 3',5'-guanosine monophosphate (cGMP) in erythropoietin (Epo) production. Further studies have been carried out to clarify the role of NO in the hypoxic regulation of Epo production in Epo producing human hepatocellular carcinoma (Hep3B) cells, which produce Epo in response to physiological stimuli. Our reverse transcriptase-polymerase chain reaction (RT-PCR) technique revealed the expression of iNOS mRNA in Hep3B cells after incubation under hypoxic (1% O2) conditions for 6 hr. Hypoxia also significantly increased medium levels of nitrite in Hep3B cells. In order to investigate the role of NO in Epo production in Hep3B cells under normoxic (20% O2) conditions, we have studied the effects of interferon-gamma (IFN-gamma) on Epo production. IFN-gamma is known to induce iNOS and enhance the production of NO. IFN-gamma produced significant increases in medium levels of Epo and nitrite. IFN-gamma also significantly increased cGMP levels in Hep3B cells. Furthermore, NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, significantly decreased IFN-gamma induced elevations in medium levels of Epo and nitrite as well as cGMP levels in Hep3B cells. These results provide further support for an important role of the NO/cGMP system in hypoxic regulation of Epo production in Hep3B cells.

Trichloroethylene health risk assessment: a new and improved process.

Trichloroethylene (TCE), an environmental contaminant of National concern, is the focus of a new health risk assessment process incorporating the Proposed Cancer Risk Assessment Guidelines. This paper describes not only how TCE became an environmental problem for the Air Force, but also details the new Risk Assessment process envisioned by the Environmental Protection Agency's (EPA) National Center for Environmental Assessment (NCEA). Insights on epidemiological evaluations, both past and future, and their impact on the cancer classification of TCE are discussed. Examples of how physiologically based pharmacokinetics and dose-response characterization described in the new Cancer Guidelines are applied to TCE are provided. In addition, a variety of modeling techniques are discussed for the development of reference doses (oral exposure) and reference concentrations (inhalation exposures) for TCE. Finally, the role of risk communication is included. This new process provides an example of how interagency (EPA, Department of Defense. Department of Energy) and extramural (industry, academia) partnerships can provide greater gains to the nation, as a whole, than any of the parts on their own.

The role of mouse intestinal microflora in the metabolism of trichloroethylene, an in vivo study.

1. Both trichloroethylene and its metabolite, dichloroacetic acid, produce liver tumors peroxisome proliferation and other adverse cellular alterations in rodents. 2. The hepatic mechanism by which dichloroacetic acid is formed is not conclusively demonstrated, but pharmacokinetic models have successfully associated its formation with trichloroacetic acid as immediate precursor. 3. Previous investigations have shown that dichloroacetic acid is formed from trichloroacetic acid by gut microflora isolated in vitro. 4. To determine the impact of gut microflora on dichloroacetic acid formation from a trichloroethylene dose in vivo, we developed a procedure which reduced gut microflora some 3 orders of magnitude below published levels. 5. The administration of trichloroethylene to control mice and to mice whose gut was practically sterile resulted in equivalent concentrations of dichloroacetic acid and other metabolites in blood and liver, but significantly different content of these metabolites in cecum contents. 6. These data indicate that gut microflora contribute minimally, if at all, to the formation of circulating dichloroacetic acid under these conditions.

Pharmacokinetic analysis of chloral hydrate and its metabolism in B6C3F1 mice.

Chloral hydrate (CH) and its metabolites, trichloroacetate (TCA) and dichloroacetate (DCA), have been shown to induce liver tumors in male B6C3F1 mice. The pharmacokinetics of CH and its metabolites play an important role in its toxicity. This study was designed to characterize the kinetics of CH metabolism, and the formation and elimination of TCA, DCA, trichloroethanol (TCOH), and trichloroethanol glucuronide (TCOG) in male B6C3F1 mice. Mice were dosed with 67.8, 678, and 2034 micromol/kg of CH through the tail vein. At selected time points, mice were killed, and blood and liver samples were collected. Samples were assayed by GC for CH, TCOH, TCOG, TCA, and DCA concentrations. After intravenous administration, CH rapidly disappeared from blood with a terminal half-life ranging from 5 to 24 min. Systemic clearance decreased from 36.0 to 7.6 liters/kg-hr with increasing CH dose, demonstrating dose-dependent pharmacokinetics. TCOH, TCOG, TCA, and DCA were detected over the study period. Formation and metabolism of CH metabolites seemed to be dose-dependent. The terminal half-lives of TCOH and TCOG were similar, ranging from 0.2 to 0.7 hr. TCA and DCA were formed rapidly from the metabolism of CH and cleared slowly from systemic circulation. The area under the blood concentration-time curve for DCA was 10-20% of that for TCA. Both TCA and DCA were slowly eliminated from systemic circulation. The concentration-time profile of DCA seemed to be driven by the blood concentration of TCA, suggesting the possibility of DCA formation from TCA metabolism.

In vivo/in vitro comparison of the pharmacokinetics and pharmacodynamics of 3,3',4,4'-tetrachlorobiphenyl (PCB77).

The rat hepatoma cell line, H4IIE, serves as a useful tool to assess potential biological effects such as induction of cytochrome P4501A1 expression. The objectives of this study were twofold: to investigate the kinetic time course and dosimetry of PCB77 in rat hepatoma cells dosed with PCB77 and in liver of rats given i.p. doses of PCB77, and to compare in vitro and in vivo P4501A1 enzyme induction responses. For the 4-day time-course study, H4IIE cells were exposed with two doses of [14C]PCB77 (0.9 and 3 microg/plate) and harvested at 15 and 30 min, 1, 2, 4, 8, and 12 hr, and 1, 2, 3, and 4 days. PCB77-derived radioactivity was detected in the cells as early as 15 min postdosing. For the dose-response study, the cells were dosed with various concentrations of PCB77 (0.00316-5.37 microg/plate) and harvested on Day 3 since ethoxyresorufin O-deethylase (EROD) activity in vitro reached its maximum on the third day postdosing. Time-course and dose-response studies revealed that only 1-3% of the total delivered dose was found in the cells, with the remainder in the media and adhering to the culture plates. For the dose-response study in vivo, male Fischer rats were dosed with a single i.p. injection of various concentrations of PCB77 (0.1-50 mg/kg body wt) and euthanized on Day 3. PCB77-derived radioactivity and EROD induction in vivo were measured. When EROD activity and PCB77-derived radioactivity in the rat hepatoma cells and in the rat liver were compared on an equivalent weight basis, there was a significant correlation (r2 = 0.985) between them. Prior to this study, no information on quantitative dosimetry and EROD activities of PCB77 has been reported to validate the in vitro assay with in vivo data.

Hydrogen peroxide in the regulation of erythropoietin (Epo) gene expression in hepatocellular carcinoma cells.

The present studies were designed and carried out to determine if hydrogen peroxide (H2O2) is involved in the regulation of erythropoietin (Epo) gene expression and stimulation of Epo production in the hepatocellular (Hep 3B) cells. Hep 3B cells were incubated with varying concentrations of H2O2 for periods of 6 hours or 24 hours. In other experiments Hep 3B cells were incubated for 24 hours with or without increasing concentrations of catalase and in the presence of H2O2. Culture medium levels of Epo were determined and quantitation of Epo mRNA was also made. The results indicate that H2O2 increases the levels of Epo mRNA and Epo hormone production in Hep 3B cells, and that catalase, the specific scavenger of hydrogen peroxide, inhibits Epo production in these cells. Based on these findings, it is concluded that H2O2 takes part in the signal transduction mechanisms in Epo production. It is recommended that further studies be undertaken to find out the source of the hydrogen peroxide in the hepatocellular carcinoma cells.

Autocrine regulation of erythropoietin gene expression in human hepatocellular carcinoma cells.

We have previously reported that a marked increase in erythropoietin (Epo) production can be demonstrated in Hep3B cell cultures in response to hypoxia (1). In order to determine whether this increase involves an autocrine mechanism, we have investigated the effects of purified human recombinant Epo (rHuEpo) on Epo production. Purified rHuEpo (5-80 mU/ml) produced a significant increase above control levels of Epo in Hep3B cell cultures under normoxic (20% O2) conditions. Hypoxic (1% o2) incubation of Hep3B cells with rHuEpo caused an increase over control levels of EpomRNA. Hep3B cells also expressed Epo receptor (Epo-R) transcripts. Binding studies [125I]Epo revealed that Hep3B cells contain a single class of binding site (kd=2.9 nmol/L and Bmax=1760 sites/cell). Antierythropoietin receptor monoclonal antibody inhibited the rHuEpo induced elevation in medium levels of Epo and blocked [125I]-Epo binding to Hep3B cell membranes. These results demonstrate that the expression of EpomRNA may be controlled, at least in part, by an autocrine mechanism.