Preclinical Efficacy and Safety of the Novel Antidiabetic, Antiobesity MetAP2 Inhibitor ZGN-1061.

Methionine aminopeptidase 2 (MetAP2) inhibition is a promising approach to treating diabetes, obesity, and associated metabolic disorders. Beloranib, a MetAP2 inhibitor previously investigated for treatment of Prader-Willi syndrome, was associated with venous thrombotic adverse events likely resulting from drug effects on vascular endothelial cells (ECs). Here, we report the pharmacological characterization of ZGN-1061, a novel MetAP2 inhibitor being investigated for treatment of diabetes and obesity. Four weeks of subcutaneous administration of ZGN-1061 to diet-induced obese (DIO) insulin-resistant mice produced a 25% reduction in body weight, primarily due to reduced fat mass, that was comparable to beloranib. ZGN-1061 also produced improvements in metabolic parameters, including plasma glucose and insulin, and, in HepG2 cells, initiated gene changes similar to beloranib that support observed in vivo pharmacodynamics. In vitro studies in ECs demonstrated that ZGN-1061 effects on EC proliferation and coagulation proteins were greatly attenuated, or absent, relative to beloranib, due to lower intracellular drug concentrations, shorter half-life of inhibitor-bound MetAP2 complex, and reduced cellular enzyme inhibition. In dogs, ZGN-1061 was more rapidly absorbed and cleared, with a shorter half-life than beloranib. Unlike beloranib, ZGN-1061 did not increase coagulation markers in dogs, and ZGN-1061 had a greatly improved safety profile in rats relative to beloranib. In conclusion, ZGN-1061 and beloranib demonstrated similar efficacy in a mouse model of obesity, while ZGN-1061 had a markedly improved safety profile in multiple in vitro and in vivo models. The lower duration of exposure characteristic of ZGN-1061 is expected to provide a meaningfully enhanced clinical safety profile.

Idiopathic pulmonary fibrosis diagnosed concomitantly with diffuse squamous cell lung cancer on surgical lung biopsy: a case report.

BACKGROUND: Idiopathic pulmonary fibrosis is a disease with a poor prognosis and has been associated with increased lung cancer incidence. CASE PRESENTATION: We report the case of a Caucasian 75-year-old woman, a former smoker, hospitalized for breathlessness with a chest computed tomography scan showing an interstitial lung disease. A surgical lung biopsy was performed, confirming a pattern of usual interstitial pneumonia but also numerous disseminated foci of well-differentiated focally invasive squamous cell carcinoma without hypermetabolic lung nodule, mass, or enlarged lymph node visualized on chest computed tomography or positron emission tomography scan. Nintedanib was started for its antifibrotic and antitumor properties, without any other antineoplastic treatment. Three years after initiation of nintedanib, clinical, functional, and computed tomography scan evaluations were stable, and there was no evidence for evolution of the squamous cell carcinoma. CONCLUSIONS: Data are scarce regarding the benefit of nintedanib in patients with idiopathic pulmonary fibrosis-associated lung cancer, and it is unclear whether nintedanib could have a preventive role in lung carcinogenesis in idiopathic pulmonary fibrosis patients. This experience could help the scientific community in case of similar incidental findings.

Nebulization of the acidified sodium nitrite formulation attenuates acute hypoxic pulmonary vasoconstriction.

BACKGROUND: Generalized hypoxic pulmonary vasoconstriction (HPV) occurring during exposure to hypoxia is a detrimental process resulting in an increase in lung vascular resistance. Nebulization of sodium nitrite has been shown to inhibit HPV. The aim of this project was to investigate and compare the effects of nebulization of nitrite and different formulations of acidified sodium nitrite on acute HPV. METHODS: Ex vivo isolated rabbit lungs perfused with erythrocytes in Krebs-Henseleit buffer (adjusted to 10% hematocrit) and in vivo anesthetized catheterized rabbits were challenged with periods of hypoxic ventilation alternating with periods of normoxic ventilation. After baseline hypoxic challenges, vehicle, sodium nitrite or acidified sodium nitrite was delivered via nebulization. In the ex vivo model, pulmonary arterial pressure and nitric oxide concentrations in exhaled gas were monitored. Nitrite and nitrite/nitrate were measured in samples of perfusion buffer. Pulmonary arterial pressure, systemic arterial pressure, cardiac output and blood gases were monitored in the in vivo model. RESULTS: In the ex vivo model, nitrite nebulization attenuated HPV and increased nitric oxide concentrations in exhaled gas and nitrite concentrations in the perfusate. The acidified forms of sodium nitrite induced higher levels of nitric oxide in exhaled gas and had longer vasodilating effects compared to nitrite alone. All nitrite formulations increased concentrations of circulating nitrite to the same degree. In the in vivo model, inhaled nitrite inhibited HPV, while pulmonary arterial pressure, cardiac output and blood gases were not affected. All nitrite formulations had similar potency to inhibit HPV. The tested concentration of appeared tolerable. CONCLUSION: Nitrite alone and in acidified forms effectively and similarly attenuates HPV. However, acidified nitrite formulations induce a more pronounced increase in nitric oxide exhalation.

Variations in the frequencies of torque teno virus subpopulations during HAART treatment in HIV-1-coinfected patients.

Sera from 15 patients coinfected with TTV and HIV-1, collected before and at two times after introduction of highly active anti-retroviral therapy (HAART), were tested for TTV load and the presence of the five highly divergent TTV phylogenetic groups. Seven patients showed a 1-5 log TTV load decrease during HAART, while the others did not show significant variations. A decrease in the number of coinfecting TTV genogroups was detected in 12 of 15 patients, with the mean number of TTV genogroups/patient decreasing from 2.33 before HAART to 1.47 at the last collect. All five genogroups were less frequently found after introduction of HAART. Three hundred sixty-seven TTV clones from four different genogroups, derived from two patients, were sequenced. Noticeable fluctuations in TTV subpopulation frequencies were observed in both patients analyzed. In conclusion, HAART tends to reduce the number of TTV genotypes/genogroups and may affect the balance between different TTV isolates coinfecting single individuals.

Molecular epidemiology of hepatitis B virus in an isolated Afro-Brazilian community.

This study was conducted in an Afro-Brazilian, slave-descendant community with high (42.4%) hepatitis B virus (HBV) prevalence. Twenty (8.4%) out of the 239 subjects under study were HBsAg-positive, and HBV-DNA was detected in 59 (25%) individuals. A high rate (18.3%) of occult infection was therefore observed that was associated to low HBV loads (mean, 1.8 x 10(4) copies/ml) and to a specific amino acid substitution (C100Y) in the small surface antigen. Genotyping of 50 isolates showed that 43 (86%) were of subgenotype A1, one (2%) from subgenotype A2, and five (10%) from subgenotype D. Mixed genotypes A1 and E were observed in one (2%) sample. The genetic distance (0.8 +/- 0.3%) among the HBV/A1 isolates from the community was smaller than the intragroup divergence among A1 isolates from Brazil as a whole, but it was similar to that found between A2 isolates from different countries, suggesting that HBV/A1 was introduced in the community through different sources. The substitution W501R (polymerase), previously reported only in Gambia, was observed in 46% of the HBV/A1 isolates. The precore/core promoter region of HBsAg-positive isolates showed several substitutions that could explain the anti-HBe phenotype found in 18 of 20 (90%) of the HBsAg-positive subjects.

High prevalence of human Torque teno virus in streams crossing the city of Manaus, Brazilian Amazon.

AIMS: Torque teno virus (TTV) is a human DNA virus chronically infecting most healthy individuals worldwide and can be transmitted by faecal-oral route. The occurrence of TTV was evaluated in the streams crossing the city of Manaus (Brazilian Amazon) over a 1-year period, four times a year. METHODS AND RESULTS: Fifty-two water samples were collected from 13 different locations. Viruses were concentrated from two litres of water by adsorption to negative membrane filters followed by ultrafiltration. TTV DNA was detected by PCR assays designed to detect all five TTV genomic groups. By conventional PCR, 19/52 (37%) samples were positive. By real-time PCR, TTV DNA could be detected in 48/52 (92%) samples. Viral loads ranged from 1300 to 746 000 genome equivalent per 100 ml of river water. Eleven distinct nucleotide sequences were obtained. CONCLUSIONS: Our results show the wide distribution and diversity of TTV among Manaus urban micro basins. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here may contribute to substantiate TTV as a sensitive indicator of human contamination.

New human erg isoforms generated by alternative splicing are transcriptional activators.

The erg gene is a member of the ets gene family. ETS proteins have been shown to bind specifically the (GGA-A/T) motif and to transactivate via this consensus sequence. The human erg products exhibit approximately 70% homology with ETS proteins in their DNA-binding domain. We have isolated three erg cDNAs from a human fetal liver library. Two of them are different from the previously described erg-1 and erg-2 cDNAs (Rao et al., Science, 1987, 237, 635-639), in the middle of their coding sequence and in their 5' part where a novel initiation codon is introduced. These isoforms are generated by alternative RNA splicing from a single gene that leads to the inclusion or exclusion of different exon sequences. The three cDNAs expressed by an in vitro transcription-translation system direct the synthesis of proteins of approximately 38, 49 and 55 kDa. These in vitro erg products were tested for their DNA-binding activity by gel mobility-shift assays with different probes containing the ETS-specific binding site. The results indicated that all these erg isoforms are able to bind the ETS binding site in a specific manner. Our data using transient transfection assays indicate that erg protein isoforms function as transcriptional activators.

First-in-class pan caspase inhibitor developed for the treatment of liver disease.

A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of alpha-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.

Low rate of occult hepatitis B virus infection among anti-HBc positive blood donors living in a low prevalence region in Brazil.

OBJECTIVE: The aim of this study was to determine the rate of occult hepatitis B virus (HBV) infection among blood donors living in a geographic region of low (5.6%) anti-HBc prevalence. SUBJECTS AND METHODS: Sera from 150 candidate blood donors whose blood was rejected due to total anti-HBc reactivity (despite absence of HBsAg) were tested for anti-HBs and IgM anti-HBc antibodies, as well as for HBeAg/anti-HBe. Serum HBV DNA was sought by using a PCR assay able to amplify part of the surface gene. Viral load was measured in the PCR positive samples. RESULTS: The pattern 'anti-HBc alone' (without HBsAg and anti-HBs antibodies) was found in 64 (42.7%) subjects. IgM anti-HBc and anti-HBe antibodies were detected in 2 (1.3%) and 80 (53.3%) samples, respectively. No sample was HBeAg-reactive. HBV DNA was repeatedly found in five (3.3%) samples, three of which were anti-HBs positive and two anti-HBs negative. All five HBV DNA positive samples showed a low viral load (<1000copies/ml). CONCLUSIONS: The data indicated a low rate of occult infection among anti-HBc positive, HBsAg negative blood donors living in a region of low prevalence of infection. Viral load was very low in all HBV infected subjects.

A multiplex PCR assay able to simultaneously detect Torque teno virus isolates from phylogenetic groups 1 to 5.

Torque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. TTV has an extreme genetic heterogeneity which is reflected in its current classification into five main phylogenetic groups (1-5). Using specific PCR assays, it has been shown that many individuals are co-infected with TTV isolates belonging to different phylogenetic groups. Here, a multiplex PCR assay was developed, using five recombinant plasmids. Each plasmid carried an insert of different size issued from a TTV isolate belonging to a different group. The assay was able to simultaneously amplify DNAs of TTV isolates belonging to all five phylogenetic groups. Multiplex PCR was then tested satisfactorily on DNAs extracted from 55 serum samples (47 health care workers and 8 AIDS patients). All individuals but nine were infected with at least one TTV isolate. Co-infection with multiple isolates was found in 29/47 (62%) health care workers and in 8/8 (100%) AIDS patients. A number of discrepancies were observed when results obtained with three thermostable DNA polymerases were compared. For example, four TTV phylogenetic groups were detected in a particular serum sample by using one of the three DNA polymerases, whereas the other two enzymes were able to detect only three TTV groups. However, none of the three enzymes used could be broadly considered to be more efficient than the others. Despite its limitations, the assay described here constitutes a suitable tool to visualize the degree of co-infection of a given population, avoiding time-consuming experiments.

Restriction maps of human adenovirus types 2, 5, and 3 for BstEII, MluI, NdeI, NruI and SfiI endonucleases.

The positions of cleavage sites for BstEII, MluI, NdeI, NruI and SfiI restriction endonucleases in the DNA from human adenovirus (Ad) serotypes 2, 5 and 3 were determined. In addition, the sites of cleavage for BglII in Ad3 DNA were located. All these enzymes possess a narrow specificity and generated a small number of discrete DNA fragments. Ad3 DNA was not cleaved by MluI and SfiI. It was the first observation of the absence of cleavage of an adenovirus DNA by a restriction endonuclease.

Expression of the chloramphenicol acetyl transferase gene in human cells under the control of early adenovirus subgroup C promoters: effect of E1A gene products from other subgroups on gene expression.

A hierarchy of dominance has been observed in HeLa cells co-infected with two serotypes of adenovirus belonging to different subgroups. DNA replication and late protein synthesis of one serotype are inhibited by those of the other. The degree of inhibitory effect has the following decreasing order: adenovirus type 3 (Ad3) and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad12 (A), Ad2 and Ad5 (C) [Delsert and D'Halluin, Virus Res. 1 (1984) 365-380]. HeLa cells were first transfected with recombinant plasmids carrying Ad5 E2A or E3 promoters fused to the chloramphenicol acetyl transferase gene (cat), and then infected with human Ad belonging to different subgroups. All the serotypes tested were found to be able to stimulate both E2A and E3 promoters. When HeLa cells were co-transfected with either of the previous plasmids, plus a second plasmid carrying the Ad3 E1A region, the same stimulatory effect was observed. However, an inhibitory effect on Ad5 E2A and E3 promoters seemed to occur when both Ad2 E1A (subgroup C) and Ad3 E1A (subgroup B) genes were present together. To determine which one of the early products was responsible for the observed repression effect, and to assign the target on the genome of subgroup C Ad, a plasmid was constructed in which the sequences at the 5' end of the Ad2 E1A region were fused to the structural sequences of the cat gene. In HeLa cells transfected with this plasmid, CAT activity was significantly increased after co-transfection with a plasmid carrying the Ad2 E1A region, but decreased with a plasmid carrying the Ad3 E1A region.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimizing brachytherapy for locally advanced cervical cancer.

PURPOSE: No adequate high dose rate brachytherapy technique exists to cover all known tumor volume by using one type of applicator in patients presenting with a cervix carcinoma extending to the vaginal wall and the parametria. METHODS AND MATERIALS: We adapted the existing high dose rate applicator, existing of two ovoids and one intrauterine tube, to achieve adequate irradiation of the uterus, the parametria, and the vaginal wall in these patients. Using the optimization program of the Nucletron Planning System, isodose curves were obtained to apply a specified dose of 8.5 Gy at point A and at 5 mm depth of the vaginal wall by using a single applicator for both fractions. RESULTS: Fractionated high dose rate brachytherapy can be given with both higher dosimetric accuracy and more adequate irradiation of the vaginal and the parametrial tumor component after adapting the existing high dose rate applicator for brachytherapy in cervical cancer.

Simulation accuracy in radiotherapy for maxillary sinus tumors.

PURPOSE: To evaluate the accuracy and clinical importance of beam positioning during simulation of radiation treatment for tumors in the maxillary sinus. METHODS AND MATERIALS: Five patients were prepared as if they were to be treated for a maxillary sinus tumor. A three-beam computed tomography (CT) scan-based computer plan was made for each patient. The location of the central beam axis of each beam was measured, relative to bony anatomical structures. A simulation was performed using the bony references to position the radiation beams during simulation. After this, the simulation procedure was repeated by the use of a noninvasive external localization frame with a known accuracy and reproducibility within 2 mm margins. RESULTS: When defining the clinical target volume as the known tumor with a 1 cm margin, three out of five patients would suffer a partial geographical miss throughout the entire radiation treatment due to erroneous beam positioning at the simulation stage when using bony structures as a guide for beam positioning. The influence of these errors is analyzed as normal tissue complication and tumor control probabilities. CONCLUSION: When defining a planning target volume, one should consider a margin to correct for possible simulation errors. We advise the use of objective, external (and thus nonanatomical) landmarks as a reference during simulation to reduce this extra margin to a minimum. In case of simulation, using bony structures as a reference, an additional margin should be entered, depending on the simulation accuracy that can be obtained.

Measuring radiation fibrosis: the interobserver reliability of two methods of determining the degree of radiation fibrosis.

PURPOSE: To compare the interobserver reliability of the palpation method with the method of measuring tissue compliance with a tissue compliance meter (TCM) on women who underwent breast-conserving surgery and radiotherapy for breast cancer. METHODS AND MATERIALS: Thirty-eight patients and 30 controls were measured with the palpation method by two radiation oncologists and with the TCM by two physiotherapists. Measurements were taken on four locations of the breasts of all 68 women. Reliability coefficients were computed for both methods. A weighted kappa score was computed for the palpation method and this was compared with the intraclass correlation coefficient (ICC) computed for the TCM method. The conditions for direct comparison of these scores were met in this study. RESULTS: A weighted kappa of 0.65 was computed for the palpation method and an ICC of 0.91 was computed for the TCM method. These scores differ significantly from each other (p < 0.01). CONCLUSION: The interobserver reliability of the TCM method is superior to that of the palpation method. However, at locations where the TCM is not applicable, palpation is a good alternative.

Optimization of interstitial volume implants.

For interstitial applications of high dose rate (HDR) afterloading brachytherapy, generally a single stepping iridium-192 source is used, enabling optimization of the dose distribution by optimization of the relative time (dwell time) that the source remains at a certain position (dwell position). We analysed the effects of geometric optimization in a regular volume implant, with strictly parallel catheters, and in an irregular volume implant, such as an implant for tumours of the base of the tongue characterized by a non-parallel geometry and varying catheter separations. In both examples the reference dose is specified at 85% of the mean central dose (as is done in the Paris system for dose specification) in the non-optimized as well as the optimized plan. The irradiated volume, the dose uniformity, and the choice of the reference dose of optimized and non-optimized dose distributions were compared. This was done by isodose plots for representative planes, volume dose histograms (distributed, contiguous, and natural), and dose non-uniformity ratios (DNRs). For the regular implant, optimization results in a 28% increase in the treated volume with a similar increase in the overdosed volumes. In order to keep the treated volume comparable with the non-optimized dose distribution, 90-95% of the mean central dose should be chosen as a reference dose or the range of active dwell positions should be shortened in case of optimization. In the case of the irregular volume implant at the base of the tongue, the method for dose specification should be kept unchanged after geometric optimization as the volume enclosed by the reference isodose does not increase. It is clear from the volume-dose histograms that there is a reduction of the overdosed volume due to optimization. This is accompanied by an increase in the uniformity index and a decrease of the DNR. In conclusion, geometric optimization appears to be an effective tool to improve the dose distribution of interstitial volume implants. Contiguous and natural volume dose histograms appear, apart from planar dose plots, valuable methods for evaluating the dose distribution of an implant.

Low frequency of mutations in the core promoter and precore regions of hepatitis B virus in anti-HBe positive Brazilian carriers.

BACKGROUND: Mutations in the core promoter and precore regions of the hepatitis B virus (HBV) genome, notably the double substitution (AGG to TGA) at nt positions 1762-1764 in the core promoter, and the precore stop codon mutation G to A at nt 1896, can often explain the anti-HBe phenotype in chronic carriers. However, the A1896 mutation is restricted to HBV isolates that have T at nt 1858. The double substitution at positions 1762-1764 has been described to occur preferentially in patients infected with strains showing C instead of T at nt 1858. RESULTS: HBV DNAs from 29 anti-HBe Brazilian samples were characterized by nucleotide sequencing of PCR products from precore region. Among them, 18 isolates presented C at nt 1858 (mostly genotype A strains). The 11 remaining isolates (genotypes D and F) had T1858. The stop codon mutation at nt 1896 was found in seven isolates (24% of the total and 63% of the isolates that had T1858). The frequency of the double substitution at positions 1762-1764 was surprisingly low (20%) among C1858 isolates. An association between A1896 and TGA 1762-1764 mutations was observed among genotype D isolates: these showed either none of the two mutations or both. Furthermore, strains mutated at positions 1896 and/or 1762-1764 also presented an elevated number of other, less common substitutions in the core promoter and precore regions. CONCLUSIONS: The data reported here are not in accordance with some reports from other parts of the world. In half of the isolates, none of the mutations previously described could explain the anti-HBe phenotype.

Lack of evidence for an association between TTV infection and severe liver disease.

BACKGROUND: In 1997 a new human virus, TTV, was identified. The clinical significance of the TTV infection, however, remains unknown. OBJECTIVE: Establishment of the prevalence of TTV DNA in different population groups in Germany and the assessment of the possible clinical significance of TTV infection. STUDY DESIGN: Detection of the TTV DNA by PCR in blood donors, patients with end-stage liver disease, and multiple transfused patients with haemotological disorders. RESULTS: TTV DNA was detected in 16 of 122 (13.1%) volunteer blood donors, in 34 of 77 (44.2%) patients with end-stage liver disease, and in 21 of 38 (55.3%) multiple transfused patients. There was no difference in the prevalence of the TTV DNA in end-stage liver disease patients with regard to sex, age, presence of HCV and HBV infection markers, and etiology of liver disease. Phylogenetic analysis of the amplified DNA fragments from 12 randomly selected TTV infected subjects demonstrated that in Germany at least two putative TTV genotypes and four subtypes are circulating. CONCLUSIONS: (i) TTV is widely spread in German population; (ii) one of the possible ways of its transmission is blood transfusion; (iii) TTV infection most probably does not generally lead to the development of the end-stage liver disease.

Detection of hepatitis A virus RNA in serum during the window period of infection.

BACKGROUND: Hepatitis A virus (HAV) infection is the leading cause of clinically apparent viral hepatitis in many parts of the world, including developed and developing countries. Only limited information is available regarding the seronegative viremic window that follows HAV infection, and no systematic search has been reported for HAV RNA positive, IgM anti-HAV negative serum samples during hepatitis A outbreaks. OBJECTIVES: To determine the proportion of HAV infected individuals among (i) children who were tested negative for anti-HAV antibodies during hepatitis A outbreaks which occurred in a public school (n = 157) and a child care center (n = 38); (ii) subjects (n = 46) initially classified as acute non-A-C hepatitis patients after clinical examination and serological tests (sporadic cases). STUDY DESIGN: Reverse transcription (RT)-PCR was performed to detect the presence of HAV genome in serum samples collected from anti-HAV negative, susceptible subjects. RESULTS: HAV RNA was detected in 19/157 (12%) and 5/38 (13%) anti-HAV negative children from the public school and child care center, respectively. Twelve (26%) out of the 46 acute hepatitis patients (sporadic cases) were also HAV RNA positive. From nine of these 12 patients, a second blood sample was obtained 18-34 days after the first one: all nine had seroconverted to IgM anti-HAV, and their serum transaminases had reached elevated levels (mean ALT, 418; mean AST, 241). CONCLUSIONS: Detection of HAV RNA before IgM anti-HAV seroconversion may be used as an early diagnosis method during hepatitis A outbreaks. HAV RNA testing should also help to elucidate acute hepatitis cases of unknown etiology.

Modes of cell death in rat liver after monocrotaline exposure.

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) plant toxin that produces sinusoidal endothelial cell (SEC) injury, hemorrhage, fibrin deposition, and coagulative hepatic parenchymal cell (HPC) oncosis in centrilobular regions of rat livers. Cells with apoptotic morphology have been observed in the livers of animals exposed to other PAs. Whether apoptosis occurs in the livers of MCT-treated animals and whether it is required for full manifestation of pathological changes is not known. To determine this, rats were treated with 300 mg MCT/kg, and apoptosis was detected by transmission electron microscopy and the TUNEL (TdT-mediated dUTP nick end labeling) assay. MCT produced significant apoptosis in the liver by 4 h after treatment. To determine if MCT kills cultured HPCs by apoptosis, HPCs were isolated from the livers of rats and exposed to MCT. MCT caused a concentration-dependent release of alanine aminotransferase (ALT), a marker of HPC injury. Furthermore, caspase 3 was activated and TUNEL staining increased in MCT-treated HPCs. MCT-induced TUNEL staining and release of ALT into the medium were completely prevented by the pancaspase inhibitors z-VAD.fmk and IDN-7314, suggesting that MCT kills cultured HPCs by apoptosis. To determine if caspase inhibition prevents MCT-induced apoptosis in the liver, rats were cotreated with MCT and IDN-7314. IDN-7314 reduced MCT-induced TUNEL staining in the liver and release of ALT into the plasma. Morphometric analysis confirmed that IDN-7314 reduced HPC oncosis in the liver by approximately 50%. Inasmuch as HPC hypoxia occurred in the livers of MCT-treated animals, upregulation of the hypoxia-regulated cell-death factor, BNIP3 (Bcl2/adenovirus EIB 19kD-interacting protein 3), was examined. BNIP3 was increased in the livers of mice treated 24 h earlier with MCT. Results from these studies show that MCT kills cultured HPCs by apoptosis but causes both oncosis and apoptosis in the liver in vivo. Furthermore, caspase inhibition reduces both apoptosis and HPC oncosis in the liver after MCT exposure.