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Several investigations have been conducted to develop in vitro bioaccessibility (IVBA) assays that reliably predict in vivo oral relative bioavailability (RBA) of arsenic (As). This study describes a meta-regression model relating soil As RBA and IVBA that is based upon data combined from previous investigations that examined the relationship between As IVBA and RBA when IVBA was determined using an extraction of soil in 0.4 M glycine at pH 1.5. Data used to develop the model included paired IVBA and RBA estimates for 83 soils from various types of sites such as mining, smelting, and pesticide or herbicide application. The following linear regression model accounted for 87% of the observed variance in RBA (R2 = .87): RBA(%) = 0.79 × IVBA(%) + 3.0. This regression model is more robust than previously reported models because it includes a larger number of soil samples, and also accounts for variability in RBA and IVBA measurements made on samples collected from sites contaminated with different As sources and conducted in different labs that have utilized different experimental models for estimating RBA.

Mathematical approaches are not well established for calculating the upper confidence limit (UCL) of the mean of a set of concentration values that have been measured using a count-based analytical approach such as is commonly used for asbestos in air. This is because the uncertainty around the sample mean is determined not only by the authentic between-sample variation (sampling error), but also by random Poisson variation that occurs in the measurement of sample concentrations (measurement error). This report describes a computer-based application, referred to as CB-UCL, that supports the estimation of UCL values for asbestos and other count-based samples sets, with special attention to datasets with relatively small numbers of samples and relatively low counts (including datasets with all-zero count samples). Evaluation of the performance of the application with a range of test datasets indicates the application is useful for deriving UCL estimates for datasets of this type.

The concentration of lead in indoor dust is a key parameter in the Integrated Exposure Uptake Biokinetic (IEUBK) model used by the U.S. Environmental Protection Agency (USEPA) to evaluate risks to children from lead in soil. The default assumption is that the concentration of lead in indoor dust is 70% of the concentration of lead in outdoor soil. This report reviews the basis of this assumption, and compares the assumption to data obtained at mining/smelting Superfund sites in USEPA Region 8. Data for lead concentrations measured in both indoor dust and outdoor soil at a number of different properties at nine different Superfund sites were fit to a linear model (Cdust = K0 + Ksd·Csoil). Based on ordinary linear regression, values of Ksd ranged from 0.04 to 0.34. Values of Ksd estimated using a simple method to account for measurement errors yielded values from 0.04 to 0.35. These findings indicate that the concentration of lead in dust at mining/smelting sites in Region 8 is usually not as large...

In this article we summarize the results of a series of studies that measured the relative bioavailability (RBA) of lead in a variety of soil and soil-like test materials. Reference material (Pb acetate) or Pb-contaminated soils were administered orally to juvenile swine twice a day for 15 days. Blood samples were collected from each animal at multiple times during the course of the study, and samples of liver, kidney, and bone were collected at sacrifice. All samples were analyzed for Pb. We estimated the RBA of a test material by fitting mathematical models to the dose-response curves for each measurement end point and finding the ratio of doses that gave equal responses. The final RBA for a test material is the simple average of the four end point-specific RBA values. Results from 19 different test materials reveal a wide range of RBA values across different exposure materials, ranging from 6 to 105%. This variability in RBA between different samples highlights the importance of reliable RBA data to help improve risk assessments for Pb in soil. Although the RBA value for a sample depends on the relative amounts of the different chemical and physical forms of Pb present, data are not yet adequate to allow reliable quantitative predictions of RBA from chemical speciation data alone.

Exposure of children to lead in the environment was assessed at the Murray Smelter Superfund site using both a deterministic risk assessment approach, the Integrated Exposure Uptake Biokinetic (IEUBK) model, and a probabilistic approach, the Integrated Stochastic Exposure (ISE) model. When site-specific data on lead in environmental media were input as point estimates into the IEUBK model, unacceptable risks were predicted for children living within five of eight study zones. The predicted soil cleanup goal was 550 ppm. Concentration and exposure data were then input into the ISE model as probability distribution functions and a one-dimensional Monte Carlo analysis (ID MCA) was run to predict the expected distribution of exposures and blood lead values. Uncertainty surrounding these predictions was examined in a two-dimensional Monte Carlo analysis (2-D MCA). The ISE model predicted risks that were in the same rank order as those predicted by the IEUBK model, although the probability estimates of exceedin...

Community groundwater systems serve as the primary source of household water for over 81 million people in the United States. In many residences, groundwater may contain significant concentrations of radon-222, serving as a potential source of human exposure. An extensive database of measured radon concentrations in community groundwater systems is available from the National Inorganic and Radionuclides Survey (NIRS). However, estimation of key descriptive statistics and distribution functions for these data is complicated by the fact that the data are censored, that is, a portion of the NIRS samples have concentrations below the minimum reporting level of 100 pCi/L. A variety of parameter estimation techniques were investigated for fitting the NIRS data, including maximum likelihood and regression methods. In general, the NIRS data were found to be well-described by lognormal distributions. The resulting lognormal distributions, along with quantitative estimates of uncertainty, are presented for five gro...

Bioavailability of lead (Pb) has become an issue in quantifying exposure of sensitive populations and, where necessary, establishing cleanup levels for contaminated soil. Immature swine were used as a model for young children to estimate the degree to which Pb from two fully characterized composite samples from the Smuggler Mountain Superfund Site in Aspen, Colorado may be bioavailable to resident children. The composite soils contained 14,200 and 3870 micrograms Pb/g of soil. Relative and absolute enteric bioavailabilities of Pb in soil (oral dose groups of 75,225, and 675 micrograms Pb/kg body wt/day) were estimated by comparison with an orally administered soluble Pb salt (lead acetate = PbAc2.3H2O) (dose groups of 0, 75, and 225 micrograms Pb/kg body wt/day) and an intravenously administered aqueous solution of Pb (100 micrograms Pb/kg/ day) from the same trihydrate salt administered daily for 15 days to 50 juvenile swine. The biological responses (area under the blood Pb concentration-time curve, and the terminal liver-, kidney-, and bone-lead concentrations) produced by Pb from PbAc2.3H2O and lead-contaminated soils were determined. This study revealed Pb from soil containing 14,200 micrograms Pb/g of soil had a bioavailability relative to Pb from PbAc (RBA), ranging from 56% based on the area under the blood lead concentration-time curve (AUC) versus dose, to 86% based on calculations from liver-Pb loading versus dose. Similarly, Pb from soil containing 3870 micrograms Pb/g of soil had an RBA ranging from 58% based on the AUC versus dose, to 74% based on calculations from liver- and kidney-Pb loading versus dose. Bioavailability of Pb in soils may be more or less than EPA's default RBA of 60%, therefore, measuring site-specific RBAs provides a basis for improved exposure and risk assessment.

This study describes a method for measuring the relative oral bioavailability (RBA) of arsenic (As) in soil and other soil-like media using young swine as the animal model. Groups of animals are exposed to site soil or sodium arsenate orally for 12 d. Forty-eight-hour urine samples were collected from each animal on d 6-7, 8-9, and 10-11 and were analyzed for total As. The urinary excretion fraction (UEF) for each group was estimated by plotting the mass of As excreted in urine by each animal as a function of the dose administered, and then fitting a linear model to the data using simultaneous weighted linear regression. The RBA of a test material is calculated as the ratio of the UEF value for the test material divided by the UEF of the reference material. Uncertainty around the RBA estimate is calculated using Fieller's theorem. Application of this method to a series of test soils indicates that RBA values for As can range from 18 to 52%. This wide variability supports the conclusion that there may be important differences in RBA between sites, and that use of a site-specific RBA value is likely to increase the accuracy of risk estimates for exposure to As in soil.

This report summarizes the results of a study to develop an in vitro bioaccessibility (IVBA) extraction technique for estimating the relative bioavailability (RBA) of arsenic (As) in soil. The study was implemented in several steps. In step 1, key variables in the extraction protocol were identified. In step 2, 21 different extraction conditions were tested on 12 different soils with reliable RBA values measured in swine or monkeys to identify which yielded useful in vivo-in vitro correlations (IVIVC). In step 3, three extraction conditions were evaluated using 39 different test soils to make a final selection of the best IVIVC. In step 4, the within- and between-lab reproducibility of the extraction method was examined. The optimum IVIVC model for swine utilized a pH 1.5 IVBA extraction fluid, with an R (2) value of .723. For monkeys, the optimum IVIVC model was obtained using a pH 7 IVBA extraction fluid that contained phosphate, with an R (2) value of .755. Within-lab precision of IVBA results was typically less than 3%, with an average of 0.8% for all 4 labs. Between-lab variation in mean IVBA values was generally less than 7%, with an overall average of 3%. The principal advantages of this IVBA method compared to other in vitro methods described in the literature are that (1) the fluids and extraction conditions are simple, (2) the results have been calibrated against a larger data set than any other method, and (3) the method has been demonstrated to be reproducible both within and between labs.

(1996). Characterization of cancer risk associated with exposure to chloroform. Journal of Environmental Science and Health, Part C: Vol. 14, No. 2, pp. 81-104.

Monte Carlo modeling is a powerful mathematical technique that offers many advantages compared to traditional point estimate methods for characterizing the inherent variability in exposure to environmental chemicals among different members of a population. However, Monte Carlo analyses of variability are often limited by uncertainty (lack of knowledge) about the true distribution of key exposure and risk parameters. Because of this uncertainty, it is not appropriate to select only one value (either a “best estimate” or, even worse, an intentionally conservative value) for each uncertain parameter, because the result of a simulation employing such fixed point estimates is only one of many possible results that could be true. The solution to this problem is to run repeated Monte Carlo simulations, using different combinations of the uncertain parameters as inputs. The degree of variation between the output of different simulations then reveals how certain (or how uncertain) any particular estimate ...

Although small arms ranges are known to be contaminated with lead, the full extent of metal contamination has not been described, nor has the oral bioavailability of lead in these soils. In this work, soil samples from ranges with diverse geochemical backgrounds were sieved to

The effect of several halomethanes on protein synthesis has been studied in isolated hepatocytes. When cells are added to medium preequilibrated with CCl4 or CBrCl3, protein synthesis is inhibited after a lag period of 4 to 10 min. The concentrations of CBrCl3, CCl4, and CHCl3 which cause a 50% inhibition of protein synthesis are about 6 μM, 400 μM, and 4mM, respectively. This order of potency parallels the rate at which these compounds are metabolized by the hepatic mixed function oxidase, suggesting that metabolism is required for toxicity. The inhibitory effect caused by 18 min of exposure to CBrCl3 is not reversed when the toxin is removed, indicating that inhibition involves some irreversible modification of cellular material. Unexpectedly, the inhibitory effect caused by 18 min of exposure of CCl4 is about 30–40% reversed when the toxin is removed. This suggests that CCl4 causes inhibition not only by a metabolism-dependent (irreversible) pathway, but by a metabolism-independent (reversible) mechanism as well. Extracellular Ca2+ is not required for CCl4 inhibition of protein synthesis.

Isolated pituitary cells prepared from adrenalectomized rats secrete ACTH in response to corticotrophin releasing factor (CRF) contained in extracts of rat hypothalamic-median eminence tissue (HME-CRF). Neither puromycin nor cycloheximide, at doses which inhibit protein synthesis by up to 86%, significantly affect HME-CRF stimulated ACTH secretion. When pituitary cells are exposed to corticosterone, after a lag of approximately 30 min they become hyporesponsive to stimulation by HME-CRF. Graded doses of puromycin inhibit this action of the steroid hormone to the same degree that they inhibit protein synthesis. This quantitative correlation suggests that protein synthesis is an intermediate step in the mechanism of corticosterone action on the pituitary. On the other hand, cycloheximide does not block corticosterone action, even at concentrations of cycloheximide which inhibit protein synthesis by 90%. Although the basis for these divergent effects is not yet clear, several important implications are discussed.

The separation of the 12 SH central subunit, the 5 SE peripheral metallo subunit, and the 1.3 SE biotinyl carboxyl carrier protein which are formed on the dissociation of transcarboxylase has been accomplished by molecular sieving on Bio-Gel. The 12 SH and 5 SE subunits have been obtained in nearly homogeneous form as judged by the sedimentation velocity profiles and by acrylamide gel electrophoresis. The 1.3 SE carboxyl carrier protein has given less consistent results; sometimes a single band of molecular weight of approximately 12,000 is obtained on gel electrophoresis in sodium dodecyl sulfate but sometimes there is an additional band of lower molecular weight of approximately 11,000. This lower molecular weight component may result from limited proteolytic degradation in spite of efforts to prevent it. Two or more bands are obtained in the absence of dodecyl sulfate. This heterogeneity may result from aggregation. subunits. The most effective reconstitution is accomplished by a two-step process. First, the 1.3 SE carboxyl carrier protein and 5 SE metallo subunit are combined to form the 6 SE complex; then this product is combined with the 12 SH subunit to yield active enzyme. With a limiting amount of the 6 SE complex and an excess of the 12 SH subunit the resulting enzymatic activity is proportional to the concentration of 6 SE complex. Likewise, with an excess of the 6 SF, complex and the 12 SH subunit limiting, the enzymatic activity is proportional to the concentration of the 12 SH subunit. The maximum specific activities of the reconstituted 6 SE complex and the 12 SH subunit were approximately 60% and 50%, respectively of the value estimated for their specific activities in the native 18 S form of the enzyme with three peripheral 6 SE subunits. Because assay of the 6 SE complex is done using an excess of the 12 SH subunit, this may yield enzyme with a single peripheral subunit. The 6 SE subunit may be less effective in this form. In the case of the 12 SH subunit, the activity is most likely low because the reconstituted 6 SE complex does not contain the full complement of 1.3 SE biotinyl carboxyl carrier proteins. The carboxyl carrier protein provides the groups which link together the 5 SE peripheral subunits with the central subunit. Evidence is presented that the 1.3 SE biotinyl carboxyl carrier must first combine with the 5 SE subunit to assume a form which effectively provides the combining groups for association with the 12 SH subunit.

The properties of the Ca2+, Mg2+-ATPase of erythrocyte membranes from patients with cystic fibrosis (CF) were extensively compared to that of healthy controls. Following removal of an endogenous membrane inhibitor of the ATPase, activation of the enzyme by Ca2+, calmodulin, limited tryptic digestion or oleic acid, as well as inhibition by trifluoperazine, were studied. The only properties found to be significantly different (CF cells vs controls) were calmodulin-stimulated peak activity (90 vs 101, P less than 0.02) and trypsin-activated peak activity (92 vs 102, P less than 0.02). No significant difference could be measured in the steady-state Ca2+-dependent phosphorylation of CF and control erythrocyte membranes indicating similar numbers of enzyme molecules per cell. The functional state of Ca2+ homeostasis in intact erythrocytes was investigated by measuring the resting cytosolic free Ca2+ levels using quin-2. Both CF and control erythrocytes maintained cytosolic free Ca2+ between 20 to 30 nM. Addition of 50 uM trifluoperazine resulted in an increase in erythrocyte cytosolic free Ca2+ to about 50 nM in both CF and control cells. Estimates of erythrocyte membrane permeability using the steady-state uptake of 45Ca into intact erythrocytes revealed no differences between CF and control cells. These results confirm that there is a small decrease in the calmodulin-stimulated activity of the erythrocyte Ca2+, Mg2+-ATPase in CF. However, this deficit is apparently not large enough to impair the ability of the CF erythrocyte to maintain normal resting levels of cytosolic free Ca2+.

ATP-dependent calcium sequestration by rat liver microsomes has been analyzed by steady state isotope exchange. Liver microsomes display high affinity for Ca2+; the half-maximal concentration of free Ca2+ is 0.10 microM, and intravesicular steady state concentrations of 7-8 mM Ca2+ are achieved under optimal conditions. The uptake system displays multiphasic kinetics with respect to both Mg-ATP and free Mg2+, suggesting that microsomal preparations contain two distinct Ca2+-sequestering systems. Measurement of the kinetics of Ca2+ sequestration permits independent assessment of the activity of the calcium active transport system(s) and of the permeability of the membrane to Ca2+ backflux. Addition of ionophore A-23187 to microsomes renders them more permeable, and this is reflected in a more rapid equilibration of isotope. Conversely, low levels of free Ca2+ lead to a decreased rate of active transport, and this is reflected in a lower initial rate of isotope exchange. This system should be useful for investigating the mechanisms by which hormones, hepatotoxins, and other agents influence Ca2+ fluxes in cells.

Lymphocytes prepared from normal individuals and patients with cystic fibrosis (CF) were compared with regard to intracellular Ca2+ concentration, distribution, and handling. No difference between control and CF was found in the concentration of cytosolic free Ca2+ (98 +/- 5 vs 102 +/- 7 nM), and no difference was observed in the kinetics with which control and CF cells restored cytoplasmic Ca2+ toward normal following a perturbation induced by cold-exposure. However, total intracellular Ca2+ is about 25% higher in CF lymphocytes than in control. Of this excess Ca2+, about 50% appears to be sequestered in mitochondria. This suggests that some difference in Ca2+ handling does exist, but the significance of this in cystic fibrosis remains to be determined.

Two tryptic hydrolysates of bovine liver glutamate dehydrogenase which had been maleylated prior to digestion were investigated. In one of these the carboxymethylated protein was used, in the other the protein had been oxidized with performic acid. These soluble derivatives were rapidly hydrolyzed by trypsin and after approximately 5 min, no additional hydrolysis was observed. From these two digests peptides accounting for 474 of the known 506 residues of the polypeptide chain were isolated. These peptides ranged in size from 2 to 73 residues. As expected, some of these peptides contained arginine as the only basic residue, indicating that these peptides are preceded by arginyl residues. Other peptides contained from 1 to 7 lysyl residues with a single arginyl residue, which was COOH-terminal in the peptide. Such peptides supplied overlaps for the component lysyl tryptic peptides. Many peptides were isolated which contained from 2 to 4 arginyl residues as well as several lysyl residues, thus providing additional overlaps at arginyl residues. Several segments of the protein were obtained which had not been found in fragments produced by other methods of scission. In all, the peptides obtained in this investigation established or confirmed 31 of the 33 overlapping sequences involving lysyl residues and at 9 arginyl residues as well. Some evidence is presented to explain the observations of limited action of trypsin at many arginyl residues. This appears to be due to the presence, at or near the potentially susceptible bond, of either acidic residues in the sequence itself or to hydroxyl groups which had become partially or completely maleylated. This is consonant with out observations that many peptides of identical composition manifested different electrophoretic mobilities or appeared in different fractions eluted from ion exchange columns.

Halogenated hydrocarbons (CCl4, BrCCl3, 1,1-dichloroethylene, bromobenzene) cause a wide spectrum of dysfunction and injury in liver cells. An early effect of CCl4, BrCCl3, and 1,1-dichloroethylene is destruction of the Ca2+-sequestering ability of the endoplasmic reticulum, and it has been suggested that this lesion leads to subsequent disruption of other cell functions. Work to test this hypothesis has begun in this and other laboratories. While it appears that redistribution of intracellular Ca2+ does occur following these agents, the importance of this in cell injury is not fully resolved. Current results suggest Ca2+ redistribution may be involved in some cases (e.g., surface blebbing caused by bromobenzene), but not in others (e.g., inhibition of lipid secretion by CCl4).

Transcarboxylase is made up of a central hexameric subunit (S20,W similar 12 S), three peripheral dimeric metallo subunits (S20,W similar to 5 S), and six biotinyl carboxyl carrier subunits (S20,W similar to 1.3 S). The results presented here show that the carboxyl carrier subunit is required for assembly of the 12S and 5S subunits into the oligomer. However, only a portion of the subunit is required for assembly. On treatment of transcarboxylase briefly with trypsin at pH 6.3 extremely susceptible peptide bonds of the carboxyl carrier protein are cleaved releasing biotinyl peptides of about similar to 66 and similar to 40 residues. The resulting trypsinized transcarboxylase, though enzymatically inactive, remains essentially intact as judged by its hydrodynamic and molecular sieving properties. The modified enzyme can be dissociated at pH 8 to the central 12S subunit and peripheral 5S subunit to which the residual portion(s) of the cleaved carboxyl carrier protein is still attached. These components can then be separated by molecular sieving. The residual portion of the carboxyl carrier protein (non-biotinyl peptide) can then be isolated by dissociation of the 5S subunit complex at pH 9 and by chromatography over Bio-Gel A-1.5m. The isolated non-biotinyl peptide has been shown to contain the combining domain of the 1.3SE carboxyl carrier protein since it causes combination of the 12S and 5S subunits. Active enzyme is formed by combination of the intact carboxyl carrier protein and the 12S and 5S subunits and an inactive oligomer of similar size is formed if the non-biotinyl peptide is used in place of the carboxyl carrier protein. The similar to 66- and similar to 40-residue biotinyl peptides, which are released by the trypsin treatment, apparently occur on an exposed portion of the enzyme. This portion of the carboxyl carrier protein apparently serves to place the biotinyl group adjacent to the two substrate sites of the enzyme, one of which is on the peripheral subunit and the other on the central subunit. Thus the carboxyl carrier protein has two functions: one portion holds the 12S and 5S subunits in juxtaposition and the other portion orients the biotinyl group adjacent to the substrate sites so that it may function as a carboxyl carrier between the sites.

Isolated pituitary cells prepared from adrenalectomized rats secrete ACTH in response to CRF, and this response is inhibited by corticosterone. Both the stimulation of release by CRF and the inhibition of release by corticosterone are antagonized by cordycepin (3'-deoxyadenosine). Inhibition of CRF-stimulated secretion by cordycepin is apparently not related to inhibition of RNA synthesis, since high doses of actinomycin D do not affect ACTH secretion. More likely, cordycepin's inhibition of secretion stems from its inhibition of adenylate cyclase activity. Inhibition of corticosterone action by cordycepin is qualitatively similar to that previously reported for actinomycin D. This effect of both drugs is probably due to inhibition of RNA synthesis. Significantly, a low dose of cordycepin has a greater inhibitory effect on cortico-sterone action than on total cellular RNA synthesis. Cordycepin is reported to preferentially inhibit messenger RNA synthesis, and a low dose preferentially inhibits appearance of cytoplasmic RNA in pituitary cells. These data suggest that corticosterone-induced RNA is a cytoplasmic (messenger) RNA.

The rate of growth hormone (GH) biosynthesis in pituitary cells prepared from rats with different histories of glucocorticoid exposure was analyzed by a dual-labeling acrylamide-gel technique. Glucocorticoid deficiency, produced by adrenalectomy, reduces GH synthesis by approximately 50%. GH synthesis in adrenalectomized rats treated with either natural or synthetic glucocorticoids, is restored to normal or above normal levels. Acute exposure (1 h) of cells to corticosterone in vitro does not produce a significant increase in GH synthesis. Adrenalectomized rats treated with steroid for one day show a small but detectable increase in GH synthesis, while treatment for 3–6 days results in progressively larger stimulatory effects. All of these results are similar to previous findings regarding glucocorticoid stimulation of GH synthesis in vitro.

Exposure of isolated rat hepatocytes to hepatotoxic halomethanes results in a 40–60% decrease in intracellular Ca 2+ content. The order of halomethane potency (CBrCl 3 CCl 4 CHCl 3 ) suggests that this effect requires halomethane metabolism by the hepatic mixed function oxidase system. Although the Ca 2+ sequestering ability of the endoplasmic reticulum is destroyed by CBrCl 3 and CCl 4 , it appears that much of the Ca 2+ lost from the cell is mitochondrial in origin. Paradoxically, saturating concentrations of CCl 4 cause a marked increase in cell Ca 2+ . CCl 4 also causes an acute increase in cytoplasmic free Ca 2+ (from about 60 nM to about 90 nM), but this effect does not appear to require CCl 4 metabolism and is probably a result of direct action of CCl 4 on the plasma membrane.

Liver cell injury induced by carbon tetrachloride involves initially the metabolism of carbon tetrachloride to trichloromethyl free-radical by the mixed function oxidase system of the endoplasmic reticulum. It is postulated that secondary mechanisms link carbon tetrachloride metabolism to the widespread disturbances in hepatocyte function. These secondary mechanisms could involve the generation of toxic products arising directly from carbon tetrachloride metabolism or from peroxidative degeneration of membrane lipids. The possible involvement of radical species such as trichloromethyl (.CCl3), trichloromethylperoxy (.OOCCl3), and chlorine (.Cl) free radicals, as well as phosgene and aldehydic products of lipid peroxidation, as toxic intermediates is discussed. Data do not support the view that an increase in cytosolic free calcium is important in the toxic action of carbon tetrachloride or bromotrichloromethane. In addition, carbon tetrachloride-induced inhibition of very low density lipoprotein secretion by hepatocytes is not a result of elevated levels of cytosolic free calcium.

Secretion of lipid as very low density lipoprotein (VLDL) by isolated hepatocytes was studied in a system in which the cells were exposed to a constant concentration of carbon tetrachloride (CCl 4 ) throughout the duration of the incubation. Inhibition of secretion was characterized in terms of CCl 4 concentration and duration of incubation. Half-maximum inhibition of VLDL secretion occurred at 80 μM CCl 4 . At 390 μM CCl 4 , VLDL secretion was inhibited 40% in 2 min and was suppressed completely in 5 min. No CCl 4 -dependent release of cellular glutamic oxaloacetic transaminase occurred under the conditions studied. Evidence is presented indicating that the metabolism of CCl 4 is required for the expression of CCl 4 -dependent inhibition of lipid secretion. With respect to the parameters studied, the isolated hepatocyte system closely mimics the hepatic response of the intact animal to CCl 4 .

A consensus does not exist regarding the nature of mechanisms linking the initial events of CCl4 metabolism to emergence of the classical indices of CCl4, liver cell injury. The possibility that a CCl4-dependent disturbance of intra-hepatocellular calcium homeostasis might be a linking mechanism was investigated with isolated hepatocytes in suspension. CCl4-dependent inhibition of very low density lipoprotein secretion was studied. On the basis of kinetic data, dose-response data, and failure of elevated cytosolic calcium levels to inhibit lipid secretion, it was concluded that disturbed intracellular calcium homeostasis probably is not important in CCl4-dependent inhibition of secretion of very low density lipoproteins.

The objective of this work was to identify the natural substrates of cyclic AMP-dependent protein kinase in pituitary cells. Studies were performed using 2 systems: intact pituitary cells stimulated with dibutyryl cyclic AMP (DBC) after preincubation with [gamma-32P]. Phosphorylation of proteins was analyzed by two-dimensional gel electrophoresis, followed by autoradiography. In intact cells, the only clear and reproducible effect of DBC stimulation is increased phosphorylation of 3 proteins (termed A, B, and C), each with a molecular weight of about 20 000 dalton. The time-course and dose-dependence of phosphorylation of A, B and C are generally similar to that for DBC-induced hormone secretion, which is consistent with a role for these proteins in the secretory mechanism. When [gamma-32P]ATP is added to cell extracts, proteins A, B, and C are not measurably phosphorylated, either in the absence or presence of cyclic AMP. This observation suggests that proteins A, B and C may not be directly phosphorylated by cyclic AMP-dependent protein kinase, but may be phosphorylated indirectly by a second kinase. On the other hand, growth hormone and prolactin are readily phosphorylated in cell extracts by cyclic AMP-dependent protein kinase (although they are not phosphorylated in vivo). This finding makes clear the need for caution in interpreting results from broke cell systems.

SummaryACTH secretion by isolated pituitary cells is stimulated both by HME-CRF and DBC, and when given in combination, the two secretagogues interact synergistically. Although the mechanism of this interaction is unknown, the potentiating effect of DBC is displayed without an apparent time lag and persists after removal of the cyclic nucleotide. Corticosterone inhibits ACTH secretion induced by HME-CRF and DBC, acting alone or in combination. The implications of these findings are discussed.The authors are grateful to Beth Wiblin Portanova and James Roe for their expert technical assistance.

Carbon tetrachloride (CCl 4 )-induced inhibition of very low density lipoprotein (VLDL) secretion was studied in isolated hepatocytes. The hypothesis that inhibition of secretion is due to altered calcium homeostasis following CCl 4 -dependent inhibition of endoplasmic reticulum calcium sequestration was investigated. Inhibition of VLDL secretion by CCl 4 was not dependent on extracellular calcium, since inhibition occurred when extracellular calcium was reduced to 0.1 μM. CCl 4 inhibited hepatocyte VLDL secretion more rapidly than it inhibited microsomal calcium sequestration. Further, the concentration of CCl 4 that produced half-maximal inhibition of VLDL secretion was about one-half the concentration required to produce half-maximal inhibition of microsomal calcium sequestration. The calcium ionophore A23187 did not mimic the action of CCl 4 in inhibiting VLDL secretion under conditions in which A23187 altered cellular calcium homeostasis. The results suggest that an alteration of calcium homeostasis is not involved in inhibition of VLDL secretion by carbon tetrachloride.

A tentative but almost complete amino acid sequence for the subunit peptide chain of bovine liver glutamate dehydrogenase indicates a minimal size of 506 residues with a molecular weight of 56,100, in accord with the physical size of the subunit of 55,900. Inactivation with pyridoxal 5′-phosphate, followed by reduction with sodium borohydride, has permitted identification of the essential lysine as residue 97. Nitration of tyrosine-412 is accompanied by loss of the allosteric inhibitory effect of guanosine triphosphate. Comparison of the sequences of glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase has indicated that only two 12-residue sequences are similar in the two enzymes; this sequence includes reactive lysine-97 of the former enzyme.