Toxin-induced increase in survival factor receptors: modulation of the threshold for apoptosis.

The threshold at which toxins induce cell death is thought to directly relate to the amount of injury sustained. We show that the threshold at which a cell initiates toxin-induced death may vary in response to changes in the trophic environment. Treatment of Rat-1 fibroblasts with 50-175 mM dimethylformamide (DMF) induced cell death by apoptosis. Addition of insulin-like growth factor 1 (IGF-1; 100 ng/ml) and/or overexpression of the IGF-1 receptor (IGF-1R) attenuated the cytotoxicity of DMF. Furthermore, 95-99% of cells were protected from DMF-induced apoptosis if cells were pretreated with platelet-derived growth factor (5 ng/ml) for 16 h before treatment with DMF in the presence of IGF-1. Platelet-derived growth factor induced the expression of IGF-1R mRNA. The ability of cells to proliferate and survive after a 24-h treatment with DMF was determined by colony formation; whereas treatment with concentrations of >130 mM DMF reduced cellular survival, exposure to concentrations of <130 mM unexpectedly increased the colony-forming ability of treated cells when compared to that of controls. Treatment of Rat-1 fibroblasts with 75 and 130 mM DMF induced IGF-1R mRNA as determined by reverse transcription-PCR analysis. Serum withdrawal also transiently increased the expression of IGF-1R mRNA in Rat-1 fibroblasts. These results show that cells can actively adapt to pathological and physiological stress by up-regulating receptors that provide signals for cellular survival. We suggest that the threshold for toxin-induced apoptosis is determined not only by the extent of cytotoxic damage but also by the trophic environment and the ability of a cell to modulate survival signals that attenuate toxicity.

Apoptosis and cytotoxins.

Most xenobiotics ultimately become lethally cytotoxic, according to concentration. Toxins with completely disparate mechanisms of action induce apoptotic cell death. This suggests that the threshold for the onset of cell death can be determined by the relative expression levels of genes which promote or suppress apoptosis. The selectivity of a toxin may thus be determined not only by the selective imposition of perturbation or the amount of damage inflicted, but also by how readily that cell engages apoptosis. Measuring damage to cells, therefore, does not necessarily predict outcome. The threshold for apoptosis is determined not only by the static phenotype of the cell, which may confer a high or low survival potential, but also by its capability when stressed to modulate the expression of genes which control survival. The trophic environment of a cell can also influence the threshold for death. These findings have a profound impact on concepts defining toxin selectivity and on attempts to use mechanistic information to predict toxicity to organisms.

Alteration of transcription factor mRNAs during the isolation and culture of rat hepatocytes suggests the activation of a proliferative mode underlies their de-differentiation.

The commonly observed loss of liver specific phenotype regularly described in rat hepatocyte culture is typified by the loss of total cytochrome P450 (CYP) content and the altered abundance of CYP mRNAs. The current work shows that these changes are preceded by the induction of the mRNA encoding the transcription factor c-jun during the hepatocyte isolation procedure. Then as the hepatocytes attach to the substratum the induced expression of c-jun subsides and two patterns of CYP mRNA loss are observed. The mRNAs encoding CYPs 2C11, 2C13, 2E1, 3A1, 3A2 and 4A1 continuously decline while CYP 1A2, 2A1/2 and 2B1/2 mRNAs are temporarily stabilised for 2 to 2.5 hours at a reduced level before declining further. The loss of CYP1A2 and 2B1/2 mRNAs parallels the loss of the mRNAs encoding the liver specific transcription factors C/EBP alpha and HNF-1. The early and rapid increase in c-jun mRNA followed by a decline in C/EBP alpha mRNA are characteristic of the changes in the expression of these transcription factor mRNAs following the stimulation of hepatocyte proliferation after partial hepatectomy. The finding that the rate of loss of total P450 following partial hepatectomy parallels that in rat hepatocyte culture suggests that the commonly employed hepatocyte isolation procedure "primes" the normally quiescent hepatocytes to enter the cell cycle and de-differentiate especially as both systems lose the major constitutively expressed CYP2C11 isozyme.

The mechanism of hepatic uptake of a radiolabelled monoclonal antibody.

Clinical and experimental scintigraphic studies have found that radiolabelled antibodies are not only taken up by tumour(s) but also by normal liver. The accumulation of radionuclides in this organ poses a major problem to the use of radiolabelled antibodies as diagnostic and therapeutic tools. In an attempt to understand the mechanism of hepatic uptake and clearance of radiolabelled antibodies, the intrahepatic biodistribution of an 111In-labelled MAb (HMFG1), was determined following i.v. administration to normal male rats. Two hours after administration the liver contained 15% of the injected dose, with most of the remaining radioactivity in the blood. The hepatic burden of the 111In MAb remained constant over the next 72 hr in the face of decreasing blood levels of radioactivity as well as its urinary and faecal excretion. At 2 and 72 hr after injection, 50% and 10% respectively of the hepatic radiolabel was due to blood borne antibody. Following a collagenase-cell isolation procedure, only 23% of the amount remaining in the liver at 2 hr was found to be cell-associated; 66% was lost during the cell isolation and purification procedure. Cellular uptake increased with time so that, by 72 hr after administration, 58% was cell-associated and 29% freely removable. At all timepoints, the parenchymal cells contained more activity than non-parenchymal cells. No evidence of antibody-receptor interactions could be obtained either in vivo or in cultures of hepatic parenchymal and non-parenchymal cells. Our data suggest that the bulk of the hepatic burden of 111In MAb results from extravascular pooling of the antibody.

Validation of tonometric measurement of gut intramural pH during endotoxemia and mesenteric occlusion in pigs.

Tonometry is a minimally invasive method for estimating gastrointestinal intramural pH (pHi). Tissue pH is calculated by using the Henderson-Hasselbalch equation and measurements of arterial [HCO-3] and CO2 tension (PCO3) of saline contained in a Silastic balloon within the lumen of the gut. The validity of the method rests on two key assumptions: 1) PCO2 in saline in the tonometer balloon is similar to tissue PCO2 and 2) tissue and arterial [HCO-3] are similar. To validate this method, ileal pHi measured directly with a microelectrode was compared with pHi estimated tonometrically in four groups of anesthetized pigs. Group I (n = 4) were controls. In group II (n = 4), intestinal tissue acidosis was induced by total occlusion of the superior mesenteric artery (SMA). In group III (n = 5), acidosis was induced by partial occlusion of the SMA. In group IV (n = 4), tissue acidosis was induced by endotoxemia. Agreement was excellent between direct and tonometric measurements in groups I and IV and less good in groups II and III. Weighted mean correlation coefficients (rw) for the two measurement methods were 0.743 and 0.9447 in groups II and IV, respectively. Correlation coefficients for the individual animals in group III were more variable than the other groups and ranged from 0.547 to 0.990. The tonometric method for measuring GI pHi is invalid under conditions of zero flow and leads to error under conditions of low flow. However, the method is reliable in the setting of tissue acidosis induced by endotoxemia.

Quantifying changes in regional myocardial perfusion with aortic contrast echocardiography.

We developed a technique to assess regional myocardial perfusion by quantifying echocardiographic myocardial contrast appearance and intensity after aortic root injection of an agitated diatrizoate meglumine solution. The technique was validated by comparing digitized echocardiographic contrast parameters to regional perfusion in the circumflex bed determined by calibrated Doppler flow probe and antemortem monastral blue staining. Regional perfusion was altered by circumflex stenosis, occlusion, and reactive hyperemia. Contrast effects were measured in an initial subset of six dogs by peak intensity change, time to peak intensity, maximal rate of intensity rise, and mean intensity change integrated over 1, 2, or 3 seconds after contrast appearance (MI1, MI2, MI3). MI2 and MI3 best predicted regional perfusion (r = 0.93, standard error of the estimate [SEE] 0.38 ml/gm/min for each). These findings were confirmed in a second subset of six dogs (r = 0.84, SEE = 0.70 ml/gm/min). Although there was a relatively broad standard error for the prediction of absolute perfusion for the pooled data, for individual dogs data were internally consistent so that each had r greater than 0.88 for its varied flow states. The hyperemic ratio calculated by contrast echocardiography correlated well with the Doppler value (r = 0.85). Observer and study-to-study predictive variabilities were small (SEE 0.19 to 0.32 ml/gm/min). No alterations were seen in hemodynamics or reactive hyperemia after 25 consecutive injections over a 90-minute period. Contrast echocardiography with aortic root contrast injection tracks changes in regional blood flow. This approach can assess regional coronary reserve and detect changes in regional myocardial perfusion during acute ischemia and drug intervention.

Measurement of anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results.

We describe the development of a simple and highly sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for measuring IgG and IgM anticardiolipin antibodies (ACA). Microtitre plates were coated with cardiolipin at a concentration of 45 micrograms/ml by evaporation under nitrogen. Non-specific binding of diluted sera was eliminated by blocking of plates with 10% fetal calf serum in phosphate buffered saline (PBS/FCS) for 2 h. Then sera (100 microliters) at a dilution of 1:100 were incubated in the wells for 1 h. Affinity purified goat anti-human IgG or IgM (100 microliters) at a concentration of 1 microgram/ml was subsequently added and allowed to incubate for 1 h; detection of ACA was achieved using an alkaline phosphatase conjugated rabbit anti-goat IgG reagent by reading the colorimetric yield at 405 nm after incubation with substrate. Reference serum pools were established to study reproducibility of the assay throughout its sensitivity range, and Standard curves were established. The quantitative normal range was 0-9.0 Anticardiolipin ELISA Units (AEU) for IgG and 0-8.0 (AEU) for IgM-ACA. A strong correlation was found between the ELISA and radioimmunoassay methods for measuring ACA of both IgG and IgM classes. Results from 65 patients with systemic lupus erythematosus (SLE) and 45 patients with seropositive rheumatoid arthritis are also reported. The advantages of the ELISA method for quantitative determination of ACA levels, should make it a useful and reliable method for clinical and experimental monitoring of patients with SLE and associated autoimmune disorders.

[3H]thymidine-labeled colonic epithelial cells and mucosa in mice and man.

Since normal epithelial cell proliferation occurs chiefly in the lower two thirds of colonic crypts, the presence of aberrant DNA-synthesizing cells (tritiated thymidine labeled) at the mouth and on the surface of colonic crypts is being assessed as a predictive indicator of the development of neoplasia in patients at high risk. These would include patients with previous polyps or colon cancer or family history of either or both. Surface cells are obtained by pulsatile saline lavage of the lower bowel and incubated with tritiated thymidine ([3H]TdR) for autoradiographic observation. Findings in each high-risk category are presented and compared with [3H]TdR labeling data from a biopsy taken at the close of the procedure. The lavage technique has also been carried out on mice injected with the colon carcinogen 1,2-dimethylhydrazine (DMH). Mice were demonstrated to have [3H]TdR-labeled cells and cellular atypic while being hemoccult negative and asymptomatic for overt disease. Evaluation of human material preliminarily demonstrated the presence of surface-labeled epithelial cells in a high percentage of patients at risk for colon cancer.

Early detection of colonic neoplasia in patients at high risk.

An abnormal zone of DNA synthesis at the surface and upper portion of colonic crypts has been thought to be related to future adenomatous polyp development and to express a regulatory defect in the mechanism that normally terminates synthesis in the upper third. As part of a screening program for early colon cancer detection, patients over 40 years of age found to have occult blood in their stool (Ho+) are evaluated by barium enema and colonscopy as well as isotopic incorporation studies of biopsy and lavage specimens. This proliferative abnormality occurred most frequently among patients with an adenoma or adenocarcinoma although the frequency varied among simultaneous biopsies from the same patient. Specimens from Ho+ patients with a tumor often contained small areas of focal atypism in the biopsy or fragments of atypical epithelial cells in the lavage sample. A small group of Ho+ patients in whom no overt neoplasm could be detected also demonstrated surface-labeled epithelial cells with morphological alteration of these cells. Based on the microscopic findings presented, continued surveillance of these patients is suggested, as well as extension of these studies to include other high risk groups.