Cadmium affects mitotically inherited histone modification pathways in mouse embryonic stem cells.

The fetal basis of adult disease (FeBAD) theorizes that embryonic challenges initiate pathologies in adult life through epigenetic modification of gene expression. In addition, inheritance of H3K27 methylation marks, especially in vitro, is still controversial. Metals, such as Cd, are known to affect differentiation, DNA repair and epigenetic status in mES cells. We tested the premise that Cd exerts differential toxicity in mouse embryonic stem (mES) cells by targeting total histone protein (THP) production early in stem cell development, while affecting H3K27-mono-methylation (H3K27me(1)) in latter stages of differentiation. The inability of mES cells to recover from Cd insult at concentrations greater than IC50 indicates that maximum cytotoxicity occurs during initial hours of exposure. Moreover, as a measure of chromatin stability, low dose acute Cd exposure lowers THP production. The heritable effects of Cd exposure on cell proliferation, chromatin stability and transcription observed through several cell population doublings were detected only during alternate passages on days 3, 7, and 11, presumably due to slower maturation of histone methylation marks. These findings demonstrate a selective disruption of chromatin structure following acute Cd exposure, an effect not seen in developmentally mature cells. Hence, we present that acute Cd toxicity is cumulative and disrupts DNA repair, while concurrently affecting cell cycle progression, chromatin stability and transcriptional state in mES cells.

In vitro cytotoxicity testing with fluorescence-based assays in cultured human lung and dermal cells.

An in vitro study using human cultured cells was conducted to determine the reliability of fluorescence-based cell viability indicators with traditional in vitro cytotoxicity testing methods. Human lung epithelial carcinoma (A549) cells, and human embryonic skin (WS1) and lung (HFLI) fibroblasts were studied in culture to evaluate their potential to screen for cytotoxicity and to compare to previous protocols conducted in our laboratory. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and fluorescent-labeled probes were used to assess toxicity. Eight chemicals, including mercuric chloride, copper sulfate, sodium fluoride, thioridazine HCl, paraquat, amitriptyline-HCl, verapamil-HCl and chloroquine sulfate, were tested with each cell line using calcein-AM and Sytox. The data suggest that fluorescent probes are sensitive indicators of cytotoxicity and contribute to understanding the mechanisms for each chemical. In combination with previously published reports, the similarity of results among cell lines may be explained by the origin of the cell lines rather than by the diversity of the methods and indicators employed.

Subacute cytotoxicity testing with cultured human lung cells.

This study was designed to evaluate the potential of an in vitro cell culture method for its ability to determine subacute cytotoxicity and to compare the cytotoxic concentrations with rodent LD(50)s and clinical human toxicity data. Human fetal lung fibroblasts (HFL1) were incubated in the absence or presence of increasing concentrations of test chemicals for 72 h, and cell proliferation was used as a marker for toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxicity data from testing 50 chemicals, with available human lethal concentrations for the same chemicals, revealed that the 72-h experimental IC(50)s are as accurate predictors of human toxicity as equivalent toxic blood concentrations derived from rodent LD(50)s. In addition, our results demonstrate that subacute 72-h exposure of HFL1 cells more accurately predicts cytotoxicity than a 24-h mitochondrial assay previously conducted in our laboratory, although the experimental IC(50) values were not statistically different in the two assays. It is anticipated that this procedure, together with a related battery of tests, may supplement or replace currently used animal protocols to screen chemicals for human risk assessment.

Acute cytotoxicity testing with cultured human lung and dermal cells.

An extensive in vitro study with cultured cells was conducted to test the basal cytotoxicity theory. This theory suggests that most chemical injury, at least in vitro, is a manifestation of one or more insults to the basic cellular structures and functions common to mammalian cells. This accounts for the similarity of results in multilaboratory studies. Human fetal lung fibroblasts (HFL1), and human skin fibroblasts (WS1, Detroit551) were studied in culture to evaluate their potential to screen for cytotoxicity. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Twenty-nine chemicals were tested with each cell line and the cytotoxicity data compared to rodent and human lethal concentrations. The data suggest that the experimental IC50 values are as accurate predictors of human toxicity as equivalent toxic blood concentrations derived from rodent LD50s. In addition, lung and skin fibroblasts revealed no significant differences among the three cell lines. The results support the conclusion that finite cell lines of human origin have the potential for screening chemicals for human toxicity. In combination with previously published reports, the data suggest that a basal cytotoxic phenomenon may explain the similarity of results among different human cell lines.

In vitro cytotoxicity testing for prediction of acute human toxicity.

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independent in vitro cytotoxicity testing protocols, with each other and with established animal LD50 values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential of in vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD50 values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC50 values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD50s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.

In vitro cytotoxicity testing: Biological and statistical significance.

This study was designed to determine the potential of an in vitro model for predicting acute human chemical toxicity. Rat lung epithelial cells (L2) were tested for their ability to incorporate radiolabelled amino acids into newly synthesized proteins, in the absence or presence of increasing doses of the test chemical, during a 24-hr incubation. The MTT assay was also performed as a parallel measure of toxicity. IC(10), IC(50) and IC(75) values (10%, 50% and 75% inhibitory concentrations, respectively) were extrapolated from dose-response curves after linear regression analysis. The biological significance of the results of testing 30 chemicals shows that the experimental IC(50) values were more accurate predictors of human toxicity than equivalent toxic blood concentrations derived from rodent LD(50)s. Overall, the 24-hr protein synthesis experiments were at least as sensitive as the MTT protocol for detecting cytotoxicity. Individually, the toxicity of eight of 15 chemicals was underestimated with the MTT assay. In addition to calculating the correlation coefficient, the hypothesis test for B = 0 (zero slope) was computed for each experiment. This test, which is based on the slope of the sample regression equation, is used to determine the statistical significance of dose-response curves, yet it has not been routinely incorporated into cytotoxicity testing studies. It is anticipated that this procedure, together with a related battery of tests, may supplement or replace currently used animal protocols for human risk assessment.

Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts.

The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.

Temporal effects of ethanol on growth, thymidine uptake, protein and collagen production in human foetal lung fibroblasts.

Foetal Alcohol Syndrome is observed in 2/1000 live births worldwide and is characterized by decreased pre-natal and postnatal growth, physical anomalies and mental retardation. To determine the effects of ethanol (Et) on foetal cells cultured in the absence of hormones or growth factors, human foetal lung (HFL1) fibroblasts were exposed to Et-supplemented media (0.1-2% Et) for 6 hr to 7 days. Growth rates, thymidine incorporation into DNA, protein synthesis and degradation, and collagen production were assessed. For growth experiments, cells were seeded at 1 3 confluent density and incubated in Et-supplemented medium 24 hr later. Metabolic labelling was performed on confluent monolayers using [(3)H]thymidine (TdR), [(3)H]leucine or [(3)H]proline. Exposure to Et for 3 or 7 days decreased cell numbers but normal proliferation resumed when cells were re-plated in control medium. Exposure to 0.5% Et for 7 days resulted in a 3.5-fold increase in [(3)H]TdR uptake. Et suppressed protein production and enhanced degradation. The most significant decrease was seen at 6 hr, but was influenced by the amino acid used for labelling. Agarose-gel chromatography suggests that Et preferentially alters the lower-molecular-weight species. The percentage of the total protein secreted into the medium was not changed. Collagen production, as a percentage of total protein, decreased after a 48-hr label and a 7-day incubation with Et. The percentage of total collagen that was secreted into the medium was also not influenced by Et. The results indicate that, in the absence of endocrine or nutritional manipulation, acute exposure to Et in vitro inhibits cell growth and protein production; protein secretion, however, remains intact.

Effects of puromycin and hydroxynorvaline on net production and intracellular degradation of collagen in human fetal lung fibroblasts.

Amino acid substitutions in collagen that impair folding of the triple helix result in significant increases in intracellular degradation of newly synthesized collagen. We have studied the effects of agents that cause other kinds of defects in collagen: hydroxynorvaline, a threonine analog that interferes with association of pro-alpha chains; and puromycin, an antibiotic that causes premature release of nascent polypeptides. cis-Hydroxyproline and cycloheximide, whose effects on collagen synthesis and degradation have already been studied and reported, were employed as reference compounds. Human fetal lung fibroblasts were used in these experiments. All the agents inhibited total protein production, and all except cycloheximide inhibited percentage collagen production. Intracellular collagen degradation was increased in cultures exposed to puromycin, hydroxynorvaline, and cis-hydroxyproline, but not in cultures exposed to cycloheximide. These results suggest that pro-alpha chains that were either unassociated (due to hydroxynorvaline) or shortened (due to puromycin) were recognized as abnormal and degraded to the same extent as chains that contained cis-hydroxyproline. However, the increases in degradation could not account completely for the decreases in collagen production (except when cis-hydroxyproline was used at low concentrations). These findings indicate that, in addition to rendering newly synthesized procollagen molecules or partial polypeptide chains more susceptible to intracellular degradation, puromycin, hydroxynorvaline, and cis-hydroxyproline significantly inhibited collagen synthesis.

Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts.

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.

A crisis triage rating scale. Brief dispositional assessment of patients at risk for hospitalization.

The authors have developed a brief rating scale to expedite the rapid screening of emergency psychiatric patients who require hospital admission from those who are suitable for outpatient crisis intervention treatment. The interviewers used this scale to assess and score the patient rapidly on the basis of three factors: dangerousness, support system, and motivation or ability to cooperate. The authors report on and discuss the use of the scale in a preliminary study of 300 cases and in a prospective study of 122 patients who were followed for 6 months after they were evaluated. They found that those who scored below a median point on the scale required hospitalization and those who scored higher were suitable for crisis intervention as outpatients.